1 Introduction

Recently, people’s growing awareness of healthy and quality foods has increased the demand for the consumption of functional foods with numerous health benefits provided by bioactive compounds, as well as research into the development of new functional foods (Baker et al. 2022). In addition, interest in products with high bioactive compounds is evident in operational government initiatives like Biofort, HarvestPlus-China, Ayurvet Research Foundation, and HarvestPlus, which aim to enrich essential foods such as wheat with, for example, minerals (Fortuna et al. 2018; Kalra et al. 2018). Bioactive compounds that are generally regarded as safe (GRAS) (Banwo et al. 2021) are synthesized via secondary metabolism, and their yield can be influenced by different factors such as geographical, agronomic, genetic, and environmental conditions (Pinto et al. 2022). Among various approaches used to promote the production of bioactive compounds in plants as well as the growth of plants, the elicitation strategy surpasses as a robust and non-toxic technique for increasing their synthesis by modifying metabolic pathways under stress conditions (Zlotek et al. 2019; Toro et al. 2021). Additionally, due to consumers’ concerns with transgenic crops, the elicitation technique gained popularity as a method of improving the health-promoting content of plants. Moreover, the improvement of bioactive compounds by using elicitors is cheaper and more acceptable in society (Gawlik-Dziki et al. 2016). Recently, various authors also stated that the utilization of elicitors enhances the synthesis of secondary metabolites such as carotenoids and polyphenols, with their reactive oxygen species (ROS) scavenging abilities (Saini et al. 2014; Halder et al. 2019; Humbal and Pathak 2023; Moreno-Escamilla et al. 2020).

Nanoparticles (NPs), including metal-based, non-metal oxide, and carbon-based NPs, have been reported to function as potent elicitors, enhancing the generation of bioactive compounds and alleviating the adverse effects of abiotic stresses. Hence, the use of NPs as potential elicitors provides an option instead of using other chemical compounds with ecotoxicity (Mubeen et al. 2022; Vahide et al. 2021). However, the toxicity of NPs is of utmost importance in the utilization of NPs as an elicitor, as well as other properties of NPs such as cost, chemical and mineralogic composition, size, and concentration (Lala 2021).

Thus far, only a couple of NPs such as silver, gold, zinc, copper/copper oxide, etc. have been investigated as effective elicitors of plant secondary metabolites, while the functions of many other NPs including carbon dots, fullerene, graphene/graphene oxide, etc. have remained unexplored (Anjum et al. 2019). Carbon dots (CDs) are new photoluminescent NPs and rising stars of the carbon-nanoparticles family with their unique and intriguing characteristics including heavy metal-free, environmentally friendly, low toxicity, water-solubility, and good biocompatibility, etc. that outperform classic fluorescent chromophores such as tiny organic molecules and quantum dots. (Dinç et al. 2022; Baruah and Sahu 2022). Many investigations and reports based on in vitro tests revealed that among NPs CDs are still less hazardous than most bioactive NPs (Sahu et al. 2012; Liu and Tang 2020). Due to their extraordinary properties, CDs appear to be a potential candidate for an elicitor.

Despite their low-toxicity, the application of CDs to crops raises concerns about potential human and environmental risks (Guo et al. 2022b). Therefore, the selection of biocompatible, eco-friendly, green CDs synthesized from bio-safe sources using green synthesis methods is of utmost importance in agriculture applications. In this context, biomass-based carbon sources are the most promising CDs precursors because biocompatible, cheap, eco-friendly, and multifunctional CDs can easily be produced from various biomass sources (Meng et al. 2019). In addition, the synthesis of CDs from biomass sources enables the conversion of low-value biomass into value-added CDs (Kang et al. 2020; Zhu et al. 2020). Furthermore, the intrinsic heteroatom composition of biomass makes it simple to synthesize CDs doped with heteroatoms.

It is interesting to note that CDs are formed during the heating of food and food wastes, as in bottom-up techniques. Endogenous CDs have previously been isolated from sugar beet molasses, instant coffee, bread, caramel, drinks, and baked meat (Dinç et al. 2022). Recently, we reported endogenous water-soluble CDs derived from sugar beet molasses, a by-product of sugar factories, via an easy, green, and simple extraction method, as biocompatible with mammalian macrophage and fibroblast cells (Yavuz et al. 2019). In addition, lately, we reported that CDs derived from sugar beet molasses enhanced the growth of tobacco plants by mitigating the destructive effects of salt and drought stresses (Kara et al. 2023). Considering the favorable properties of these green-synthesized, renewable, and biocompatible endogenous sugar beet molasses carbon dots, we aimed to investigate the availability of these NPs as elicitors and explore their effects on the bioactive compounds of plants as well. Wheatgrass was chosen as a model plant due to its growing popularity over the last two decades and its substantial content of bioactive compounds such as minerals, phenolic compounds, and vitamins, etc. (Kaur et al. 2021). Furthermore, wheatgrass juice possesses antioxidant, antimutagenic, and anticancer activity and it has been shown to improve the treatment of patients suffering from thalassemia and anemia, and certain forms of cancer (Kalra et al. 2018; Eissa et al. 2020; Fortuna et al. 2018; Grubisic et al. 2022; Wangcharoen and Phimphilai 2016). As such, we hypothesized that utilization of sugar beet molasses carbon dots (SBM-CDs), as a novel elicitor agent, could enhance bioactive compounds (phenolics, vitamin E, C, and β-carotene, chlorophyll, and minerals), sugars, amino acids, proteins, and agronomic aspects of wheatgrass juice obtained from wheatgrass cultivated via hydroponic technique.

2 Materials and Methods

2.1 Materials

Bejostaja 1 variety of wheat (Triticum aestivum L.) was kindly procured from the Bahri Dagdas International Agricultural Research Institute in Konya. The ozone-generating equipment was from Mert Klima in İzmir. The recirculation engine was from Akyol Commercial in Ankara. The wheatgrass squeezer was from the Dermokozmetik company in Ankara, Turkey. All chemical agents used for the analyses were obtained from Merck except amino acid standards (Thermo Fisher Scientific), chlorophyll a, and phenolic compounds (Sigma-Aldrich). A high purity standard mixture of 17 amino acids supplied by Thermo Fisher Scientific (lot no: WE315610) containing L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-proline, L-glycine, L-alanine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, and L-arginine (each 2.5 ± 0.1 µMol/mL), except cystine at 1.25 ± 0.1 µMol/mL designed for compositional amino acid analysis by HPLC, was used. All HPLC eluents and phenolic compounds were HPLC grade.

2.2 Extraction of Endogenous Carbon Dots from Sugar Beet Molasses

The extraction of SBM-CDs was performed according to our previous method, where carbon dots were extracted from sugar beet molasses, a by-product of sugar factories, via a simple and green method without using any additional chemicals. Briefly, 5 g of SBM was homogenized in 10 mL of deionized water and centrifuged at 5000 rpm for 5 min. Then, the supernatant was collected and filtered through a 0.45 μm pore-size membrane filter. The filtrate was referred to as the stock solution. The solutions used in the study were prepared from this stock solution by diluting it with water. In-depth information on characterization can be obtained from our prior studies (Yavuz et al. 2019; Dinç 2016).

2.3 2.3. Cultivation of Wheatgrass and Obtaining of Wheatgrass Juice

The cultivation of wheatgrass treated with sugar beet molasses carbon dots (SBM-CDs) was carried out in the hydroponic climatization chamber of the Çumra School of Applied Sciences at Selcuk University in Konya, Turkey. Plastic trays (30 × 50 × 7 cm) were used for germination. Sterilization of the climatization chamber was performed using ozone generator equipment. The disinfection of wheat seeds was implemented by keeping seeds in a 1% sodium hypochlorite solution for 30 min, and subsequently, the seeds were rinsed with water.

Wheatgrass growth trials were carried out based on a completely randomized design using three replicates. The wheat seeds were planted in a continuous hydroponic system equipped with a recirculating system with an irrigation frequency of 20 s/120 min. Ebb and flow irrigation strategies were followed. Four different concentrations of SBM-CDs (50, 100, 150, and 200 mg L− 1) were added to the half-strength Hoagland nutrient solution to be used for the watering of wheatgrasses (Hoagland and Arnon 1950). A half-strength Hoagland nutrient solution without SBM-CDs was used as a control. Wheat grains were seeded at a density of 4 kg m− 2 with a 7-day growing period at 24 °C and 5000 lx of light intensity. The uniform-sized and shaped wheatgrasses were harvested at the end of the 7th day of germination. 7th -day-old wheatgrasses were cut 1 cm above the tray bottom with a grass clipper to be squeezed using a wheatgrass squeezer (Healthy Juicer, Lexen, California, USA) equipped with a sieve.

Wheatgrass samples were analyzed for a variety of parameters, including biomass and grass yield (g m− 2), plant length (cm), root length (cm), biomass dry matter ratio (%), grass dry matter ratio (%), and grass juice yield (g m− 2) according to (Karaşahin 2017).

Wheatgrass juice was obtained at room temperature and kept without the use of any water or chemical preservatives. The obtained wheatgrass juice samples were stored at -20 °C until analysis. The wheatgrass juice samples underwent analysis following thawing at + 4 °C.

2.4 Analysis of Proximate Composition

The pH of wheatgrass juices was determined in triplicate using a digital pH meter (Mettler Toledo). The protein contents of wheatgrass juice were analyzed according to the Dumas method using a Gerhardt Dumatherm DT Ar/He Basic (Gerhardt GmbH & Co. KG, Königswinter, Germany) (AOAC 992.23). A 100–150 mg sample was weighed in foil, and the sample was compacted. Then it is inserted into the instrument, and the results are calculated by multiplying the N content by a nitrogen conversion factor of 6.25 (Rybicka et al. 2024).

2.5 Determination of Chlorophyll a

0.1–0.2 g of wheatgrass juice is dissolved in 15 mL of acetone (80%) and properly homogenized for pigment extraction prior to absorbance measurements. The extracted liquid was filtered through a 0.45 μm filter (Arnon 1949). Spectrophotometric measurements were carried out with a 2mL filtered solution in 1 cm quartz cuvettes using a Shimadzu UV-1800 spectrophotometer at 663 and 645 nm wavelengths. 80% acetone was used as a blank in the spectrophotometer. The chlorophyll a content of wheatgrass juice was calculated using the following equation:

$${\rm{Chlorophyll}}\,{\rm{a}}\,{\rm{ = }}\,\left( {{\rm{11}}{\rm{.75x}}\,{\rm{A663}}\,{\rm{-}}\,{\rm{2}}{\rm{.35x}}\,{\rm{A645}}} \right)\,{\rm{x}}\,{\rm{20 /}}{{\rm{m}}_{{\rm{juice}}}}$$
(1)

where A663 and A645 are the absorbances of wheatgrass juice at 663 and 645 nm, respectively, and mjuice is the weight of wheatgrass juice (Fidan 2007).

2.6 Determination of Phenolic Compounds

Among the phenolic compounds, amygdalin, gallic acid, 4-hydroxy benzoic acid, vanillic acid, syringic acid, coumaric acid, rutin, benzoic acid, rosmarinic acid, and quercetin were analyzed. Analysis was carried out in an HPLC system combined with a PDA detector (Waters, Alliance 2695-2489UV) and an ACE 5 C18 (250 mm x 4.6 mm x 5 μm) column at a flow rate of 1.2 ml/min and gradient conditions at 280 nm wavelength at 25 °C. Mobile phase A was an aqueous solution of 2% acetic acid (A), mobile phase B was an equal mixture of 0.5% acetic acid and acetonitrile, and mobile phase C was acetonitrile. The gradient conditions were: A (95%) - B(5%) from 0 to 5 min; A (95 − 80%)– B (5–20%) from 5 to 8 min; and A (80 − 78%) - B (20–22%) from 8 to 10 min; A (78 − 75%) - B (22–25%) from 10 to 17 min; A (75 − 73%) - B (25–27%) from 17 to 19 min; A (73 − 60%) - B (27–40%) from 19 to 30 min; A (60 − 55%) - B (40–45%) from 30 to 35 min, A (55 − 35%) - B (45–65%) from 35 to 40 min, A (35 − 0%) - B (65 − 10%)- C (90%) from 40 to 45 min, A (0–95%) - B (10 − 0%)– C (100%) from 45 to 50 min, A (95%) - B (5%) from 50 to 62 min. The standard solutions of phenolic compounds were prepared in the methanol-water (1:1) mixture.

For the extraction of phenolic compounds, 5 mL of wheatgrass juice was homogenized with 50 mL of methanol. After adding approximately 45 mL of distilled water and thorough mixing, the solution was kept cool. The volume was then adjusted to 100 mL using distilled water. 20–30 mL of the suspension was filtered through filter paper and collected in a container, discarding the first 10 mL of filtrate. The remainder of the filtrate was transferred to a syringe and filtered through a 0.45-um filter into vials.

The calibration curve was plotted using the 1-100 mg L− 1 standard concentration range. The limit of quantification (LOQ) was in the range of 0.5-1 mg L− 1.

2.7 Amino Acid Profile

The amino acid profile of wheatgrass juices was analyzed by an HPLC (Waters, Alliance 2695) system outfitted with a fluorescence detector and AccQ Tag column (3.9 × 150 mm). The amino acids were detected at an excitation wavelength (λEx) of 250 nm and an emission wavelength (λEm) of 395 nm. Separation was performed at a 1 mL min-1 flow rate under gradient conditions with eluents of A (AccQ•Tag Eluent) and B (Acetonitrile 60%) at 37 °C. The gradient elution timeline is presented in Table 1. Before derivatization, the proteins of wheat were hydrolyzed with 6 N HCl at 120 °C for 24 h. Aspartic acid, alanine, proline, tyrosine, leucine, glutamic acid, valine, glycine, arginine, isoleucine, lysine, and phenylalanine amino acids were detected with the preparation of their standard solutions in the concentration range of 0.0125–0.15 µg L-1 via plotting the calibration curve. LOQ values were in the range of 0.25–0.75 µg L-1.

25 mg of wheatgrass juice samples were hydrolyzed in 5 mL of 6 N HCl at 120 °C for 24 h before drying. Then, the dried samples were dissolved in 5 mL of 0.2 N HCl, and the final volume was adjusted to 100 mL with distilled water. Derivatization was performed using 20 µl of this solution.

Table 1 The HPLC gradient elution program of amino acids of wheatgrass juice
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2.8 Analysis of Vitamin C, E, and β-Carotene

Vitamin E, C, and β-carotene were analyzed in an HPLC system (Waters, Alliance 2695-2489UV) coupled with a PDA detector and ACE 5 C18 (250 mm x 4.6 mm x 5 μm) column at 25 ºC. The flow rate for vitamins E and β-carotene was 1.5 mL min− 1. The separation was based on isocratic elution with an eluent of aqueous methanol (98.5%) solution. Vitamin E and β-carotene standards were prepared in concentration ranges of 1–25 mg L− 1 by dissolving with HCl and then diluted with ethanol before injection, and the injection volume was 50 µL. The detection of vitamin E and β-carotene was carried out at 326 and 285 nm wavelengths, respectively. LOQ values of vitamin E and β-carotene were 1 and 1.02 mg L− 1, respectively. Wheatgrass juices (5 mL) were initially dissolved in HCl (20 mL, 1 N) and subjected to ultrasonic treatment at 65 °C for 20 min for the extraction of vitamin E and β-carotene. Subsequently, an ethanol-hexane mixture (1:2 v/v) was added. After phase separation, 5 mL of the upper phase was diluted with ethanol (50 mL) and filtered through a 0.45 μm filter into vials for injection into HPLC. Vitamin C analysis was carried out using the method used for the determination of phenolic compounds. Quantification was carried out using standard solutions of vitamin C in the concentration range of 1-1000 mg L− 1. The LOQ value was 0.91 mg L− 1.

2.9 Analysis of Sugars

Glucose, fructose, and sucrose quantifications were performed using a refractive index detector coupled with an HPLC system (Waters, Alliance 2695). Separation was carried out on a carbohydrate column (300 × 3.9 mm; GL Sciences Inertsil NH2 5 μm, 4.6 × 250 mm), at a flow rate of 1.4 mL/min at 35 °C. Aqueous acetonitrile (75%) solution was used as the mobile phase. The injection volume was set at 20 µl. Quantitative analyses were implemented with the calibration curve plotted using sugar solutions at concentration ranges of 2–50 mg L− 1. LOQ value ​​was 3–6 mg L− 1. After dilution of grass juices (5 g) with water (40 mL), methanol (25 mL) was added and mixed thoroughly, and then; the final volume was adjusted to 100 mL with distilled water. Following filtration with a 0.45 μm membrane filter, it was injected into HPLC.

2.10 Determination of Mineral Composition

Macro (Na, K, Mg, and Ca) and microelements (Fe, Zn, Se, and Mn) of wheatgrass juices were measured on an ICP-OES device (Perkin Elmer, Optima 2100 DW). Before quantification by ICP-OES, samples were digested by acid-assisted microwave radiation. For this purpose, 8 mL of 69% (v/v) HNO3 was added to vessels containing 1 g of samples. The vessels were closed and transferred to a microwave oven adjusted to 160 °C for 30 min for decomposition. Following the digestion and subsequent cooling, the resultant solutions were diluted up to 50 mL with ultrapure water and analyzed by ICP-OES for quantification. Blank solutions were also run in each sample batch. All analyses were carried out in triplicate.

2.11 Statistical Analysis

All data were displayed as the mean ± standard error (n = 3). Statistical analyses were accomplished using the one-way analysis of variance (ANOVA) with the JMP-7 program. When ANOVA resulted in a significant F value, the differences between means were separated by the Tukey-Kramer HSD test.

3 Results

3.1 The Effect of Sugar Beet Molasses Carbon Dots (sbm-cds) on Wheatgrass Growth Indices

Biomass, wheatgrass, and wheatgrass juice yields (Fig. 1) and plant height values (Fig. 2) were significant at low concentrations (50 and 100 mg L− 1) but were low at high concentrations (150 and 200 mg L− 1) of SBM-CDs (p < 0.01), indicating that when the concentration of SBM-CDs exceeds a certain value the growth of wheatgrass is restricted. SBM-CDs treatments (150 and 200 mg L− 1) led to a statistically significant increase in wheatgrass and biomass dry matter values (p < 0.01) (Fig. 3). Compared to the control, biomass dry matter of plants subjected to 150 and 200 mg L− 1 SBM-CDs increased by 55% and 58%, respectively. On the other hand, wheatgrass dry matter at 150 and 200 mg L− 1 SBM-CDs levels increased by 26% and 29%, respectively (Fig. 3). The increase in the root lengths at 100 and 150 mg L− 1 SBM-CDs concentration levels was statistically significant (p < 0.01) (Fig. 4).

Fig. 1
figure 1

The effect of sugar beet molasses carbon dots (SBM-CDs) on biomass yield, wheatgrass yield, wheatgrass juice yield. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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Fig. 2
figure 2

The effect of sugar beet molasses carbon dots (SBM-CDs) on plant height. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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Fig. 3
figure 3

The effect of sugar beet molasses carbon dots (SBM-CDs) on biomass dry matter, wheatgrass dry matter. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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Fig. 4
figure 4

The effect of sugar beet molasses carbon dots (SBM-CDs) on root length. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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3.2 Proximate Composition of Wheatgrass Juices Treated with SBM-CDs

No significant changes were found in the pH of wheatgrass juices exposed to SBM-CDs compared to the control. The pH of wheatgrass juices varied from 5.14 to 5.98 (Table 2). The high doses of SBM-CDs (150 and 200 mg L− 1) significantly enhanced (p < 0.01) the protein contents of wheatgrass juices. 150 mg L− 1 SBM-CDs level increased protein content by 14% compared to the control sample. However, no significant difference in protein content was found at low doses of SBM-CDs (50 and 100 mg L− 1) compared to control (Table 2).

Table 2 Proximate composition of wheatgrass juices treated with SBM-CDs
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3.3 The Effect of Elicitor SBM-CDs on the Bioactive Compounds, Amino Acids, and Sugar Composition of Wheatgrass Juice

The utilization of SBM-CDs at 200 mg L− 1 concentration significantly increased (p < 0.01) the amount of β-carotene, vitamin E, and vitamin C by 2.5, 2, and 1.3 times, respectively, compared to the control (Figs. 5 and 6). At 150 mg L− 1 SBM-CDs application level, while vitamin E increased, vitamin C and β-carotene quantities statistically remained in the same group as the control group.150–200 mg L− 1 doses of SBM-CDs elicitation significantly increased (p < 0.01) the chlorophyll a values of wheatgrass juices twofold.

Fig. 5
figure 5

The effect of sugar beet molasses carbon dots (SBM-CDs) on Vitamin C and Vitamin E content. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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Fig. 6
figure 6

The effect of sugar beet molasses carbon dots (SBM-CDs) on β-carotene and Chlorophyll a content of wheatgrass juices. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01

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In general, the utilization of SBM-CDs as an elicitor significantly increased (p < 0.01) the total phenolic constituents in a dose-dependent manner compared to the control (Fig. 7). The largest quantities of amygdalin, 4-hydroxy benzoic acid, syringic acid, rosmarinic acid, and quercetin were obtained at 100 mg L− 1 of SBM-CDs concentration ratios, while 150 mg L− 1 SBM-CDs concentration favored the accumulation of vanillic acid, p-coumaric acid, and rutin (p < 0.01). The quantity of benzoic acid didn’t change at 50, 150, and 200 mg L− 1 SBM-CDs application doses compared to control. The effect of SBM-CDs on gallic acid was statistically not significant. The induction of the total content of phenolic compositions in the SBM-CDs (200 mg L− 1) treated wheatgrasses was found to be 34% higher than that of the control (Fig. 7). It can be stated that the application of SBM-CDs on hydroponically cultivated wheatgrasses can be an effective way of elicitation to enhance the phenolic compounds of wheatgrass juices.

Fig. 7
figure 7

The effect of sugar beet molasses carbon dots (SBM-CDs) on the phenolic composition of wheatgrass juices. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01. Ns: Not significant

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In this study, thirteen amino acids (alanine, tyrosine, aspartic acid, valine, proline, glutamic acid, glycine, leucine, arginine, lysine, isoleucine, and phenylalanine) were screened to test the effect of SBM-CDs on the amino acid composition of wheatgrass juices (Fig. 8). The utilization of high doses (150 and 200 mg L− 1) of SBM-CDs significantly increased (p < 0.01) the total amount of amino acids. However, the total content of amino acids at 50 and 100 mg L− 1 SBM-CDs ratios decreased with respect to the control. The most abundant amino acid in wheatgrass juice was aspartic acid and its quantity wasn’t affected by SBM-CDs treatment. The largest amount of lysine was recorded in samples exposed to 200 mg L− 1 SBM-CDs, but it was statistically not significant. The highest amounts of serine and arginine were recorded at 150 mg L− 1 SBM-CDs (p < 0.01). Glutamic acid was the amino acid determined at higher concentrations at 50 and 200 mg L− 1 SBM-CDs. Glutamic acid and proline ratios increased by 110% and 43%, respectively at 200 mg L− 1 SBM-CDs. Among aromatic amino acids, the content of phenylalanine didn’t change with SBM-CDs application except at 100 mg L− 1 SBM-CDs level, whereas tyrosine content was positively affected (p < 0.01) with SBM-CDs treatments of 150 and 200 mg L− 1. Glycine quantity decreased with SBM-CDs treatments of 50, 100, and 150 mg L− 1. Overall, 150 mg L− 1 SBM-CDs level was the one that promoted the synthesis of total amino acids of wheatgrass at the highest degree (15% increase as compared to control) (Fig. 8).

Fig. 8
figure 8

The effect of sugar beet molasses carbon dots (SBM-CDs) on the amino acid composition of wheatgrass juices. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01 and p < 0.05. Ns: Not significant

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Sucrose was found below the limit of the quantification value, so it is not demonstrated in Fig. 9. The higher concentrations of SBM-CDs (150 and 200 mg L− 1) statistically increased (p < 0.01) the fructose content of wheatgrass juices. However, SBM-CDs treatments didn’t affect glucose levels (Fig. 9).

Fig. 9
figure 9

Sugar composition of wheatgrass juices obtained from wheatgrasses treated with SBM-CDs. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01. Ns: Not significant

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The utilization of high concentrations of SBM-CDs (150–200 mg L− 1) increased the amounts of K, Mg, and Fe significantly (p < 0.01) as shown in Fig. 10. Among tested minerals, K was present in the highest amount followed by Ca and Mg in wheatgrass juices. K and Mg contents increased with SBM-CDs treatments in a dose-dependent manner as in chlorophyll contents of wheatgrass juice (Fig. 6). 50 mg L− 1 SBM-CDs decreased the amount of Ca compared to control (p < 0.05). Mn, Na, and Se amounts did not change with the application of SBM-CDs (Fig. 10).

Fig. 10
figure 10

Effect of SBM-CDs on the mineral composition of wheatgrass juices. Values are the means ± SD (n = 3). Different letters indicate the significance based on the Tukey-Kramer HSD test at p < 0.01 and p < 0.05. Ns: Not significant

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4 Discussion

Among carbon-based NPs, carbon dots have been demonstrated to possess many favorable effects on plant growth, and biotic/abiotic stresses as well (Tripathi and Sarkar 2022). Although various reports demonstrated the positive effects of CDs on plant growth such as alleviating stresses, membrane damage, etc. (Chen et al. 2016; Tripathi and Sarkar 2014; Wang et al. 2018, 2019a), only a few studies have considered the effects of CDs derived from biomass waste via an easy, simple, environmentally friendly route on the growth and nutritional parameters of plants. In this study, endogenous CDs were easily extracted from sugar beet molasses, a by-product of sugar factories, without using any passivation or chemical agents during the extraction process, which is also reported in our previous research (Yavuz et al. 2019; Dinç 2016). CDs can be supplied to the plants via foliar spraying, hydroponic, and soil cultivation methods (Li et al. 2020; Guo et al. 2022a). However, it remains unclear, and there is no consistent outcome about which application method is the most efficacious. In contrast to conventional agriculture, hydroponics facilitates the cultivation of plants without soil by using only nutrient solutions (Kalra et al. 2018). Furthermore, hydroponics offers the benefit of nutrient manipulation and enables the accumulation of some key nutrients, such as secondary metabolites, in various plants (Aires 2018; Mubeen B 2021). In this study, we used the hydroponic technique in the cultivation of wheatgrass using CDs extracted from sugar beet molasses at different concentrations (0, 50, 100, 150, and 200 mg L− 1). Biomass yield, grass yield, plant height, root length, biomass dry matter, grass dry matter ratio, and grass juice yield values were recorded on the seventh day of the wheatgrass growing period. Higher dry matter in wheatgrass could be attributed to a higher cell division rate with CDs elicitation (Vahide et al. 2021). The positive effects of SBM-CDs on plant growth and development may be linked to their ability to readily overcome biological barriers in plants to transfer more water and nutrients to promote the growth of wheat plants, which were well-correlated with the findings of Triphathi and Sarkar (2014). Similarly, CDs synthesized from the Salvia miltiorrhiza plant were also found to increase the biomass of Italian lettuce grown at 25, 35, and 45 °C (Wang et al. 2021). The ability of CDs to absorb UV light, capture light, promote electron transfer in the photosystem, enhance photosynthetic activity, and then stimulate the progression of the carbon cycle to accumulate more biomass (Guo et al. 2022b). Furthermore, the abundance of H2O binding sites in CDs promotes the growth of seedlings by absorbing more water and nutrients and thereby stimulating nutritional assimilation. Higher biomass, wheatgrass, and wheatgrass juice yields and plant height values at low concentrations of SBM-CDs show the concentration-dependent influence of CDs on plant growth. The concentration-dependent effect of CDs on biomass was in accordance with that of Lactuca sativa and mung bean sprouts reported by Hu et al.(2022a) and Wang et al.(2018) respectively. Although the molecular mechanism underlying the effects of CDs on plant growth remains unknown, it is reported that lower concentrations of CDs exhibit less aggregation and disproportionally larger absorption than higher concentrations (Hu et al. 2022a; Yan et al. 2021; Chen et al. 2016).

The enhanced protein levels of wheatgrass with the treatment of SBM-CDs could be attributed to an increased level of Rubisco enzyme, which accounts for nearly 50% of soluble proteins in many plants (Hu et al. 2022a). In addition, Rubisco, an important enzyme in the Calvin cycle, catalyzes CO2 fixation in dark reactions and facilitates the fixation of CO2 to sugar, which leads to the formation of photosynthetic outputs of carbohydrates and protein (Hu et al. 2022b). A recent study by Hu et al. (Hu et al. 2022b) whereby carbon dots were implemented in coriander (Coriandrum sativum L.) displayed an increase in protein content by 27.1% which is higher than that of our finding (Hu et al. 2022a).

Although previous studies have demonstrated the effect of elicitors on various factors, including growth parameters, phenolics, antioxidant potential, etc. of wheatgrasses/juices, the comprehensive research on the effect of biomass-derived CDs on bioactive compounds as well as nutritional quality and growing parameters of wheatgrass is still limited, and there are still knowledge gaps, like whether CDs can improve the nutritional quality of crops and what mechanisms are involved, etc. (Hu et al. 2022b). Thus, this is the first study to demonstrate CDs as a biomass-derived biocompatible elicitor that considerably improved the vitamin E, C, and β-carotene content of wheatgrass juices obtained from hydroponically cultivated wheatgrass. In accordance with our findings, Hu et al.(Hu et al. 2022b) reported that 30 and 40 mg L− 1 CDs derived from citric acid increased the vitamin C content of coriander leaves by 22.8 and 26.0%, respectively. The increase in vitamin C was attributed to the enhancement of photosynthesis upon treatment with CDs (Hu et al. 2022b). On the other hand, the vitamin C content of hydroponically cultivated Lactuca sativa L. was not affected by the CDs synthesized from rapeseed pollen explored by Zheng et al. (Zheng et al. 2017a). CDs could positively modulate carbohydrate metabolism (Yang et al. 2022) and carbohydrate metabolism supplies upstream precursors for the biosynthesis of vitamins (Li et al. 2021b).

Chlorophyll is the main (70% of the total components) and most active constituent of wheatgrass and plays a key role in the inhibition of metabolic carcinogens. Besides, as an essential pigment for photosynthetic processes, chlorophyll converts light energy into chemical energy, and its content is proportional to the rate of photosynthesis. The utilization of CDs could increase chlorophyll synthesis by inducing the overexpression of chlorophyll-related genes and the activity of the Rubisco enzyme efficiently (Wang et al. 2018; Kaur et al. 2021; Guo et al. 2022b). The chlorophyll a content of wheatgrass juice was higher than that of previous reports tested on different plants. Wang et al. (Wang et al. 2018) demonstrated an increase of up to 14.8% in the chlorophyll content of mung bean sprouts treated with CDs derived from graphite compared to the control. Similarly, commercially obtained CDs were found to enhance the photosynthesis of lettuce plants by enhancing the chlorophyll content, photosystem II performance, chloroplast activity, and rubisco activity. The increase in photosystem II was attributed to enhanced electron transfer rates by CDs (Hu et al. 2022a). Excellent light harvesting capacity CDs enable them to be used as artificial antennas to absorb UV light that plants cannot absorb, which could be used to ameliorate the photosynthesis efficiency of plants (Milenkovic et al. 2021).

Phenolic compounds, a class of plant secondary metabolites with known antioxidant properties, are one of the intriguing components of wheatgrass, possessing protective effects against oxidative stress and reducing the risk of disorders and degenerative diseases (Zlotek et al. 2019; Kaur et al. 2021). It is well known that plants increase or initiate de novo secondary metabolite biosynthesis in response to diverse biotic and abiotic stress conditions. NPs can induce ROS production, which may trigger the induction of secondary metabolism in plant systems that can be used to stimulate the production and accumulation of secondary metabolites and to protect the plant from biotic and abiotic stresses (Mubeen et al. 2022; Lala 2021). Various studies have shown that abiotic stress induced by NPs as elicitors leads to the accumulation of valuable secondary metabolites (Mubeen et al. 2022). Silver nanoparticles (AgNPs) are the most frequently used elicitors for inducing secondary metabolism in plants. However, the high cost of noble metal NPs such as silver, gold limits their commercial applications (Lala 2021). Therefore, carbon-based NPs such as CDs, particularly those derived from biomass/waste may be good alternative elicitors for the stimulation of secondary metabolites, including phenolic compounds. Thus, in this study, the profile of phenolic compounds of amygdalin, gallic acid, coumaric acid, vanillic acid, 4-hydroxy benzoic acid, syringic acid, rutin, benzoic acid, rosmarinic acid, and quercetin was analyzed after treatment with SBM-CDs. Elicitors can affect a wide range of cellular functions at the biochemical and molecular levels through inducing the upregulation of the genes. The stimulation of the synthesis of bioactive secondary metabolites, including phenolic compounds, via SBM-CDs elicitation could be linked to the enhancement of the transcriptional expression of biosynthetic genes involved in the related pathway (Elsayed S. A & Sayed S. A 2021, Vahide et al. 2021). There is no data regarding the elicitation effect of carbon dots on wheatgrass juice. However, in a newly published study by Abu Salha et al., it is reported that nitrogen-doped CDs-treated strawberry plants showed an increase in secondary metabolites (phenolics) compared to control plants (Abu Salha et al. 2023). The increase in the accumulation of phenolic compounds in wheatgrass juice upon treatment with SBM-CDs, as an elicitor, may be attributed to the up-regulation of phenylalanine ammonia-lyase (PAL) activity, which is the first enzyme of the phenylpropanoid biosynthesis pathway and contributes to the synthesis of phytoalexins and phenolics (Chandra et al. 2015). However, further comprehensive analyses and related mechanisms of SBM-CDs on the secondary metabolism of wheatgrass at the gene level are required for the clarification of the process.

Amino acids serve as precursors for a wide range of primary and secondary metabolites. In recent decades, the significance of amino acids in plant growth and stress defense has become apparent, gaining popularity in basic and applied plant science (Trovato et al. 2021). Therefore, recently, considerable efforts have been given to fortifying of essential amino acids and boosting nutrition in plants (Yang et al. 2020). Although the presence of some well-defined reports on the effects of elicitation on the synthesis of secondary metabolites in different plants, there are no reports focusing on the effects of elicitation on the amino acid composition of wheatgrass. The quantity of aspartic acid corresponds with the results obtained by Poscic et al. who reported that the aspartic acid level of barley was unchanged at 500 mg kg− 1 of cerium and titanium oxide NPs (Poscic et al. 2016). Aspartic acid is used as a nitrogen source to promote plant growth as well as a metal chelator to stabilize contaminants in soil (Wang et al. 2019b). Contrary to our lysine values, Poscic et al. found an increase in the lysine content of barley exposed to cerium and titanium oxide NPs (Poscic et al. 2016). In this study, we found a significant increase (p < 0.01) in glutamic acid and proline with the application of 200 mg L− 1 SBM-CDs. However, foliar spraying of citric acid-derived CDs (5 mg L− 1) on soybean plants was reported to improve glutamic acid and proline levels only by 6.6% and 16.5%, respectively, as recently reported by Ji et al. (2023). Even though other tested amino acids are not among the essential ones, they possess significant importance for human health. Moreover, a deficiency of non-essential amino acids directly impacts the quality and nutritional value of wheat grains (Wang et al. 2019b). Among them, the increase of proline, a strong non-enzymatic antioxidant in the plant defense system and an excellent osmolyte, in the 100, 150, and 200 mg L− 1 SBM-CDs group was remarkable (p < 0.01) compared to the control. The reason why proline accumulates at this concentration may be due to a proline decrease in proline oxidation and increased proline biosynthesis from protease or glutamate (Gohari et al. 2021b). Among aromatic amino acids, tyrosine acts as a precursor for a variety of secondary metabolites and was positively affected by certain doses of SBM-CDs (Hu and Zhou 2014). To our knowledge, there are no recent studies concerning the effects of carbon dots and their potential effects on the amino acid composition of wheatgrass juice. Gohari et al. revealed an increase in the proline content of grapevine plants upon treatment with putrescine functionalized carbon dots (Gohari et al. 2021a). In their study, the rest of the amino acids were not investigated. As a result, in our study, amino acids responded differently to SBM-CDs treatments implying the presence of certain plant responses to SBM-CDs which may require further research to elucidate their specific mechanism. The increase in amino acid contents of plants treated with CDs treatment may be attributed to enhanced N uptake and metabolism. Thereafter, enhanced uptake and transport of N leads plants to assimilate inorganic nitrogen from the atmosphere and convert it into organic nitrogen molecules like amino acids (Ji et al. 2023). Since amino acids are basic units of proteins, their increase with SBM-CDs treatment at 150 mg L− 1 naturally increased the protein content of wheatgrass juice, as shown in Table 2. In addition, the effect of amino acid increment at 150 mg L− 1 SBM-CDs level also resulted in an increase in biomass and wheatgrass dry matter values, as depicted in Fig. 2 which indicates the role of amino acids in plant growth and development.

Sugars are at the heart of the primary metabolism of plants. (Patrick et al. 2013). As endogenous soluble sugar is associated with the regeneration mechanism of plants, it is critical to consider whether carbon dots affect the soluble sugar content (Wang et al. 2021). Until now, there hasn’t been enough data to display the effects of biomass-derived carbon dots on the sugar composition of wheatgrass juice. Within the scope of this study, the amounts of fructose, glucose, and sucrose in wheatgrass juices were determined to investigate the effect of SBM-CDs. In agreement with the findings of our study, Wang et al. reported an increase in the soluble sugars (the compositions were not reported) with CDs treatments in Italian lettuce grown at 25, 35, and 45 °C compared to the control. They reported that carbon dots increased the soluble sugars in plants exposed to heat stress by effectively reducing the activity of acid invertase, sucrose degrading, and sucrose synthase enzymes (Wang et al. 2021). Correspondingly, in the study investigating the regulation mechanisms of carbon dots in the development of tomatoes and lettuce, it was reported that CDs remarkably increased the sugar content of these plants (Kou et al. 2021). CDs exposure in plants enhances the Rubisco activity and boosts the synthesis of carbohydrates via enhanced CO2 assimilation (Hu et al. 2022b) may explain the increased sugar content of wheatgrass exposed to SBM-CDs. Hence, our findings possess novelty in terms of investigating the effect of SBM-CDs as elicitor on the sugar composition of wheatgrass juices.

Wheatgrass juice is frequently used as a natural dietary supplement because of its high content of minerals (Ca, K, Fe, P, Mg, Zn, Mo, and B) and other nutrients (Grubišić et al. 2022). Considering the studies indicating that CDs treatments increase mineral uptake in plants, we postulated that elicitation of wheatgrass would enhance the mineral content of wheatgrass juices. Eight minerals, K, Mg, Ca, Na, Fe, Zn, Mn, and Se were estimated in wheatgrass juices obtained from wheatgrass hydroponically cultivated and elicited with SBM-CDs (Fig. 10). Our results showed that SBM-CDs application increased the Mg content, which is one of the key components of the chlorophyll molecule. Both Mg and chlorophyll quantities increased with high concentrations of SBM-CDs. The correlation of Mg with chlorophyll may be due to the presence of Mg in the center of the porphyrin ring of the chlorophyll. Chlorophyll has a key role in photosynthesis, carbohydrate accumulation, and stress resistance. In addition, Mg content is frequently considered as one of the fundamental tools controlling plant photosynthetic rate. Thus, Mg has a direct impact on the yield and quality of plants because of its complex role in plant physiology (Li et al. 2021a, 2023; Ghumman et al. 2017). According to our findings, the quantity of Mn remained unchanged, but that of Zn increased. Mn has been linked to the superoxide dismutase (antioxidant enzyme), whereas Zn has been linked to the activation of various plant enzyme systems. In addition, Zn inhibits heavy metal-induced oxidation of proteins and lipids (Ghumman et al. 2017). Overall, our mineral composition results agree with those of Li et al. who reported that the application of CDs increased the uptake and diffusion of K+, Ca+ 2, Mg+ 2, Cu+ 2, Zn+ 2, Mn+ 2, and Fe+ 3 in Arabidopsis thaliana (Li et al. 2019). Likewise, Hu et al. found that 40 mg L− 1 CDs concentration increased potassium content by 64.3%, Ca by 21%, Mg by 26.2%, P by 12.8%, Mn by 56%, and Fe content by 125% in Coriandrum sativum L. compared to the control (Hu et al. 2022b). Zheng et al. reported that carbon dots from bee pollen significantly increased potassium (K) absorption in Brassica parachinensis L. grown hydroponically. The increased nutrient retention capacity may be attributed to the hydrophilic groups like carboxyl and hydroxyl on the surface of CDs facilitating binding sites for water molecules and allow water to be transported into the plant. These hydrophilic functional groups over CDs, facilitating the adsorption of numerous minerals such as Ca, Cu, Zn, K, Mg, and Fe, which are vital for plant growth (Zheng et al. 2017b).

5 Conclusions

In this study, we successfully demonstrated, for the first time, the availability of biomass-derived carbon dots derived from sugar beet molasses as an elicitor. Our results highlight their favorable impacts on both agronomic aspects and the bioactive compounds of wheatgrass juice obtained from hydroponically cultivated wheatgrasses. Remarkably, the introduction of endogenous, food-bornesugar beet molasses carbon dots increased biomass dry matter values, protein contents, vitamin E, chlorophyll a, amino acids, phenolic compounds, fructose, K, Mg, and Fe in a concentration-dependent manner.

Based on our results, the utilization of environmentally friendly and biocompatible carbon dots as an elicitor can offer intriguing opportunities for both improvement in the agronomic indices and boosting nutritional quality, including bioactive compounds, amino acids, and sugars. Besides, their usage in modern agricultural systems will make a great contribution to the production of functional foods. Further, future studies should be targeted for the overproduction of specific desired bioactive components and for producing them at a commercial level by testing various parameters such as the mode of application (hydroponics, foliar, and soil), treatment in a selected period of growth, dose optimization, field-like conditions, their potential effect on other plants and fruits, etc.