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. 2023 Feb 28;13(3):104. doi: 10.1007/s13205-023-03516-z

IAA-decorated CuO nanocarriers significantly improve Chickpea growth by increasing antioxidative activities

Saad Hanif 1, Rabia Javed 2,✉, Aisha Khan 1, Anila Sajjad 1, Muhammad Zia 1,✉
PMCID: PMC9975142  PMID: 36875960

Abstract

Plant growth regulators tagged on metallic oxide nanoparticles (NPs) may function as nanofertilizers with reduced toxicity of NPs. CuO NPs were synthesized to function as nanocarriers of Indole-3-acetic acid (IAA). Powder X-ray diffraction (XRD) and scanning electron microscopy (SEM) revealed 30.4 nm size of NPs and sheet-like structure, respectively, of CuO-IAA NPs. Fourier-transform infrared spectroscopy (FTIR) confirmed CuO-IAA formation. IAA-decorated CuO NPs enhanced the physiological parameters of Chickpea plants, i.e., root length, shoot length, and biomass compared to naked CuO NPs. The variation in physiological response was due to change of phytochemical contents in plants. Phenolic content increased up to 17.98 and 18.13 µgGAE/mg DW at 20 and 40 mg/L of CuO-IAA NPs, respectively. However, significant decrease in antioxidant enzymes’ activity was recorded compared to control. Presence of CuO-IAA NPs increased the reducing potential of plants at higher concentration of NPs, while decrease in total antioxidant response was observed. This study concludes that IAA conjugation to CuO NPs reduces toxicity of NPs. Furthermore, NPs can be explored as nanocarriers for plant modulators and slow release in future studies.

Keywords: CuO NPs, Chickpea, Phytohormone IAA, Nanofertilizer, Antioxidative activity

Introduction

Nanotechnology deals with designing, production, characterization and application of nanomaterials in different fields (Nazir et al. 2021). These materials are being used in many fields like electronics, medicine, pharmacy, energy, and environmental science (Elango et al. 2015; Madhumitha et al. 2016). However, use of nanotechnology in agriculture is under-explored area (Helmy et al. 2016). Nanotechnology can be used to overcome world’s perilous developmental problems and to strengthen the economic growth and stability.

Worldwide, agriculture sector has many challenges by the use of agro-chemicals because the use of chemicals leads to environmental pollution, imbalanced ecosystem, and other ecological impacts. There is need for precision in agricultural practices to use nano-based technologies to overcome such problems (Javed et al. 2023). Currently, many materials are being used as plant growth stimulants and against pathogenic diseases (Husen and Siddiqui 2014; Faizan et al. 2021). In this perspective, use of metal oxide nanoparticles (NPs) has been reported for plants’ supplementation and growth (Das et al. 2015; Ashfaq et al. 2017; Javed et al. 2017a; Ahmad et al. 2020). Even with positive advantages of nanomaterials as growth enhancers, their sudden release, ineffective dispersion, and accumulation during use are of wide concern. Therefore, surfactant stabilization and surface functionalization during fabrication have been developed (Hasan et al. 2020; Javed et al. 2020). Hence, there is need for effective nanocarriers for the efficacious delivery of nutrients to crop plants. Moreover, apart from the favorable effects, few NPs also pose toxicity to plants, humans, and environment.

CuO NPs have diverse biotechnological applications due to their excellent redox potential, higher stability, re-producibility of nanomaterial, etc. These characteristics are due to physicochemical properties assigned to CuO NPs during synthesis procedure (Javed et al. 2017b; Naz et al. 2020; Asghar et al. 2022). These NPs have revealed remarkable antioxidant, antibacterial, sensing, and catalytic activities (Cuong et al. 2022). Moreover, CuO NPs are one of the highly toxic metallic oxide NPs demonstrated by various reports on plants (Chang et al. 2012). The positive response regarding crop growth and protection of CuO NPs on different crop plants like Spinacea oleracea and Solanum lycopersicum has been observed (Giannousi et al. 2012; Wang et al. 2019). Similarly, their negative influence has also been noticed in few studies. For instance, Costa and Sharma (2016) studied the influence of CuO NPs on Oryza sativa and observed decrease in germination rate, root and shoot length, and biomass by the increased uptake of CuO NPs in roots and shoots. In another study, CuO NPs mitigated root and shoot length as well as biomass, reduced photosynthetic rate, and elevated superoxide dismutase and ascorbate peroxidase production when applied in different Hordeum vulgare varieties (Petrova et al. 2021).

Metals play an important role in the life cycle of plants. Among those metals, copper (Cu) is considered as essential micronutrient for plants (Zia et al. 2021). Cu participates as cofactor of electron transport chain in chloroplast and mitochondrion, and an important component of regulatory proteins like plastocyanin. Cu is also used (as cofactor) by the proteins involved in ROS protection, cell wall lignification, formation of pollens, metabolism of carbohydrate, and formation of phenols in response to attack of pathogens (Printz et al. 2016). Among plant growth hormones, auxin is involved in controlling almost every aspect of plant growth (Grossmann 2010; Zhao 2010). Indole-3-acetic acid (IAA) is the most common and crucial hormone produced by fungi, plants, and bacteria. In plants, IAA has major role in the division of cells, fruit development, and elongation. It is also involved in initiation of roots, flowers and leaves. Mainly, in dicot plants, IAA prompts lateral roots and in monocots, it promotes development of adventitious roots (McSteen 2010; Fu et al. 2015).

Round the year, cultivation of crops is depleting essential metals from soil. In many areas, Cu depletion has resulted in poor plant growth and low yield. Application of Cu-based nanofertilizers is one of the solutions avoiding Cu toxicity by direct application. At the same time, toxicity of nanomaterials is also a big concern. Since CuO NPs are highly toxic in nature, these have been used in our study. However, this toxicity can be overcome by the use of mitigants, i.e., plant growth regulators. To approve or disapprove the hypothesis that IAA overcomes toxic effects of CuO NPs, this study was designed to synthesize CuO NPs decorated with IAA under the slow releasing concept and NPs’ toxicity mitigation. Cicer arietinum (Chickpea) is legume belonging to family Fabaceae. Recently, the effects of different metallic oxides NPs has been studied on Chickpea like ZnO, FeO, and TiO2 (Burman et al. 2013; Mohammadi et al. 2013; Naskar et al. 2020). However, the impact of CuO NPs on the physiological and biochemical parameters of Chickpea, especially while using these NPs as carriers of IAA under in vitro conditions has not been reported as yet. Therefore, in our investigation, the CuO and IAA nanocomplex was applied to Chickpea for growth analysis under in vitro conditions. Seedling development, plant morphology, biomass accumulation, phenolic and flavonoid content, and stress response were the major concerns of our study.

Materials and methods

Materials

Indole-3-acetic acid (IAA), NaOH, copper acetate monohydrate (Cu (CH3COO).H2O; 90%), Glacial acetic acid; and others used in this study were purchased from Merck (Germany) while Cicer arietinum (Chickpea) was purchased from local market.

Methodology

A flowchart is given in Fig. 1 showing the details about the methodology used in our study.

Fig. 1.

Fig. 1

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The general scheme of study. Copper oxide nanoparticles (CuO NPs) were synthesized by chemical reduction method, while IAA-doped CuO NPs (CuO-IAA NPs) were prepared by conjugating Cu with IAA and then reduction by alkali. The NPs were applied to Chickpea seed by filter paper bridge method. The plants were analyzed for physiological, phytochemical, and antioxidative response

Synthesis and characterization of nanoparticles

Synthesis of CuO nanoparticles

CuO NPs were synthesized according to the protocol reported by Javed et al. (2017b, 2021). Briefly, glacial acetic acid (2 mL) was mixed with 600 mL of 0.2 M copper acetate monohydrate (Cu (CH3COO).H2O; 90%) at 100 ℃ during continuous stirring. Freshly prepared 30 mL of 6 M NaOH solution was drop wise added until a homogeneous solution was obtained. To obtain CuO NPs, blackish solution was filtered using Whatman filter paper No. 1 and then washed thrice with ethanol and distilled water.

Synthesis of CuO-IAA nanoparticles

The surface attachment of IAA on CuO NPs was carried out by mixing the doping agent (IAA) with precursor salt in 1:1 ratio, i.e., 300 mL of 0.2 M copper acetate monohydrate (Cu (CH3COO)2.H2O; 90%) and 300 mL of 0.2 M IAA solution was mixed with 2 mL glacial acetic acid (CH3COOH) at 100 °C during continuous stirring. Freshly prepared 30 mL of 6 M NaOH solution was drop wise added until a homogeneous solution was obtained (Fig. 2). The solution was filtered using Whatman filter paper No. 1 to get CuO-IAA NPs followed by washing once with ethanol and thrice with distilled water.

Fig. 2.

Fig. 2

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The schematic presentation of synthesis of CuO-IAA NPs. Copper ions was first interacted with IAA. On addition of NaOH, IAA and Cu+2 ions make bonding with oxygen making a nanocomplex

Characterization of CuO and CuO-IAA nanoparticles

Different analytical methods including X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra, and scanning electron microscopy (SEM) were performed for the characterization of NPs. The crystalline phase of samples was identified by XRD technique using a PANalytical Empyrean system. X-ray powder diffraction was performed at room temperature using Cu Kα radiation (λ = 1.5406 Å). FTIR spectroscopy of synthesized NPs was performed using nicoletTM380 for the identification of NPs’ surface molecules. The NPs were pressed with KBr and made into pellet. The pellet was then tested for the presence of surface functional groups using FTIR spectroscopy. The analysis was performed in the range of 400–4000 cm−1 in the transmittance mode. Morphological studies of NPs were done by scanning electron microscope (SEM, TESCAN Mira3) operated at an accelerating voltage of 10 kV.

Seed germination experiment

Chickpea (Cicer arietinum) seeds of variety Balksar 2000 were purchased from Capital Seed Bank, Islamabad. The seeds of same size were washed with autoclaved distilled water and then disinfected with 0.1% mercuric chloride solution for 2 min. After that, seeds were again thoroughly washed with autoclaved distilled water to remove mercuric chloride traces from seeds.

The seeds were soaked overnight in autoclaved distilled water under dark condition. The experiment was performed in in vitro condition on filter paper bridge containing only distilled water to minimize the effect of nutrients that are present in soil or media. In filter paper bridge method, 5 mL autoclaved distilled water was taken in glass vial, and CuO NPs and CuO-IAA NPs were added at 5, 10, 20 and 40 mg/L concentration. IAA (1 and 2 mg/L) was used as positive control while autoclaved distilled water was used as negative control.

After that, 5 × 5 cm autoclaved filter paper was fixed in each glass vial keeping in view that the surface of filter paper should be at least 1 cm above the water level. Soaked Chickpea were fixed in each glass vial on filter paper. Vials were kept in dark environment till sprouting of seeds. All the vials were then transferred to the growth room at 25 + 2℃ and 16 h/8 h photoperiod. After 19 days, plants were harvested and analyzed for different parameters.

Evaluation of root and shoot parameters

After harvesting, the shoot and root length of plants was measured. The shoot and root were separated and fresh weight was measured. The plant material was kept at 37 ℃ in incubator for 3 days for drying to measure dry weight.

Biological and phytochemical screening of roots and shoots

The dried roots and shoots were crushed in pestle and mortar and 4 mg was suspended in 1 ml dimethyl sulfoxide (DMSO) in Eppendorf tubes. The tubes were incubated at room temperature for 24 h and then centrifuged at 10,000 rpm for 5 min. The supernatant was subjected to different biological assays (Javed et al. 2017c).

Total phenolic contents (TPC) were identified using the method described by Astill et al. (2001). Total flavonoid contents (TFC) were determined using the protocol previously described by Almajano et al. (2008). The free radical scavenging activity of test samples against DPPH and total antioxidant activity (TAC) of plant parts was evaluated using the protocol described by Clarke et al. (2013). Total reduction potential (TRP) of the test samples was evaluated according to the procedure of Vijayalakshmi and Ruckmani (2016).

Determination of antioxidant enzymes activities

For the determination of antioxidative enzymes activities, 0.1 g fresh roots were washed with distilled water and grinded in 1 mL phosphate buffer (pH 7.0). The mixture was centrifuged at 10,000 rpm for 10 min at 4 ºC and the supernatant was used for enzymatic assays.

Superoxide dismutase (SOD) activity was performed by the method of Beauchamp and Fridovich (1971) and peroxidase (POD) activity was performed by using the method of Vetter et al. (1958).

Statistical analysis

The experiment was randomized complete bloc design (RCBD) and the selection of concentrations of NPs was based on our experiments on other plants under in vitro conditions. The experiment to analyze the toxicity of NPs on Chickpea was performed in triplicate. Each replicate contained four vials against each treatment and in each vial single seed was inoculated. The phytochemical and biological assays were performed in triplicate and technical replicate were in duplicate. The data is reported as mean with standard deviation. Furthermore, the results were statistically analyzed using LSD at 0.05 percent probability.

Results and discussion

Characterization of CuO and CuO-IAA nanoparticles

CuO and CuO-IAA nanoparticles measured as 30 nm in size and crystalline in nature

XRD analysis of CuO NPs had diffraction peaks at 2θ = 32.5°, 36°, 38.7°, 42.3°, 48.8°, 53.7°, 58.2°, 61.8°, 66.3°, and 68°, which correspond to the (110/-111), (111), (200), (200), (020), (202), (113), (311), and (113) crystal planes, respectively (Fig. 3a). The detected diffraction reflections are comparable to JCPDS 48–1548 and are related to the monoclinic phase of CuO NPs (Ganesan et al. 2020; Dulta et al. 2022). The XRD diffraction data for CuO-IAA nanocomposite in Fig. 3b reveals the peaks at 24°, 30°, 31.6°, 32°, 36.4°, 38.6°, and 54.2° corresponding to the (021), (110), (111), (110), (111), (111), and (020) planes, respectively, that are in close line with the reported values of CuO NPs monoclinic cubic phase (JCPDS file no. 45–0937) (Praba et al. 2015). The XRD analysis depicted an average size of 30 nm for CuO NPs and 30.4 nm for CuO-IAA NPs. The XRD pattern revealed that the CuO NPs and CuO-IAA nanoconjugates are crystalline, with no additional diffraction peaks from other phases.

Fig. 3.

Fig. 3

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X-ray diffractogram of a CuO nanoparticles and b CuO-IAA nanoparticles. The definite peaks are formed at specific wavelength in a and b forming CuO NPs and CuO-IAA NPs, respectively

CuO and CuO-IAA nanoparticles illustrate irregular to sheet-like morphology

Morphology of the nanomaterials was investigated using field emission scanning electron microscope (FESEM). The SEM micrograph clearly show agglomerated and irregular shaped morphologies of CuO nanoparticles. However, in case of CuO-IAA, the NPs had sheet-like morphology (Fig. 4) where IAA molecule might join CuO NPs at both ends. Agglomeration of both types of NPs is due to non-calcination of the samples and interlining of IAA in case of CuO-IAA NPs (Wahab et al. 2013; Dulta et al. 2022). Such formation of nanocomplexes is prerequisite in slow releasing concept.

Fig. 4.

Fig. 4

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SEM micrographs of CuO and CuO-IAA NPs. A CuO NPs showed irregular shape, and B CuO-IAA NPs showed sheet-like morphology

CuO and CuO-IAA nanoparticles show the presence of N–H, C–C, O–H, C-H, C-N, Cu–O groups

FTIR is a technique used to measure the vibrational frequencies of bonds in the molecule. FTIR spectra of IAA, CuO NPs, and CuO-IAA NPs are given in Fig. 5. CuO NPs did not show any promising band in the FTIR spectrum. While the bands shown in IAA spectra match with the CuO-IAA spectrum.

Fig. 5.

Fig. 5

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FTIR spectra of IAA, CuO NPs, and CuO-IAA NPs. No sharp band is present in the spectrum of CuO NPs, but the spectra of IAA and CuO-IAA NPs illustrate many sharp bands due to the presence of different functional groups as assigned in IAA spectrum

In the FTIR spectrum of IAA, the absorbance band at 3413 cm−1 verifies the absence of free NH2 and can be attributed to indole ring (N–H) stretching. The sharp band at 1566 cm−1 as well as minor bands at 1456 and 1410 cm−1 are ascribed to C–C stretching for aromatic compounds, while a sequence of bands in the area of 1288 to 1051 cm−1 is attributed to C-O stretching. The absorption band at 927 cm−1 denote to O–H bend (Shetti and Nandibewoor 2009). The vibrational bands in CuO NPs spectra observed at 518 and 590 cm−1 indicate the stretching vibration of the Cu–O bond in monoclinic CuO NPs (Veisi et al. 2021).

In FTIR spectrum of CuO-IAA NPs, the bands at 3410 and 3385 cm−1 attribute to N–H stretching vibration in the pyrrole group (Szmigiel-Bakalarz et al. 2020). The broad band at 3131 cm−1 and 1663 cm−1 ascribe to O–H stretching (Abidi et al. 2011). The well-defined peak at 1555 cm−1 and small peaks at 1456, 1438, and 1425 cm−1 are ascribed to the C–C stretch for aromatic compounds. The absorption band at 1411 cm−1 is responsible for the CH3 asymmetrical stretching mode on the surface of CuO nanostructures (Javed et al. 2017d; Ain et al. 2018). Two sharp peaks at 1281 and 1087 cm−1 are assigned to the C-N stretching. The C-H and O–H bending vibrations for carboxylic acids are observed around 953–625 cm−1. Consequently, the metal–oxygen frequencies from 610 to 522 cm−1 recorded for CuO NPs are quite similar to those reported in the literature (Nagore et al. 2021).

CuO and CuO-IAA nanoparticles result in 75% seed germination

Sprouting of seeds was observed within 3 days of seed inoculation (Fig. 6). Seed germination was not affected except at 40 mg/L NPs where 75% germination was observed in the presence of both types of NPs. It has been reported that low concentration of CuO NPs do not effect seed germination. However, after the plumule growth, the NPs infect the tip that hinder further growth of the seeds (Zafar et al. 2017, 2020; Ain et al. 2018).

Fig. 6.

Fig. 6

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Filter paper bridge method for Chickpea seed germination showing A Sprouting of Chickpea seeds, B Chickpea plantlets after 19 days of exposure to NPs

Effects of nanoparticles on morphological characteristics of Chickpea plantlets

Increase of length, fresh weight, and dry weight of shoots at lower concentration of nanoparticles

The shoot length increased in the presence of low concentration IAA and CuO NPs. Gradual decrease in shoot length was observed by increasing the concentration of CuO-IAA NPs as compared to control (Fig. 7). Fresh weight of shoot was greater than control at 5 mg/L of CuO NPs and CuO-IAA NPs. Increase in the concentration of NPs decreased the fresh weight of shoots and the decrease was more obvious in presence of CuO-IAA NPs. The same trend was observed in case of dry weight of shoots.

Fig. 7.

Fig. 7

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Effect of CuO NPs and CuO-IAA NPs on shoot length of Chickpea plantlets after 19 days of seed germination. Values represent means ± standard errors from triplicates. Small alphabets mentioned on each bar show significant difference among mean values at p < 0.05

Lower concentrations of CuO NPs were favorable for plant growth. It has been reported that CuO at lower concentrations acts as nutrient for plants (Karlsson et al. 2009; Zafar et al. 2020). Results also show concentration dependent effect of NPs on plant growth which can possibly be due to the fact that plants can tolerate the NPs but to certain level. Exceeded concentration and long exposure can be toxic to plants in many different ways, i.e., damages transpiration, inhibits photosynthesis, and disturbs metabolism and oxidative stress. Meanwhile, increase in concentration of IAA can also negatively affect the growth, for example, Ag-IAA-treated plants showed one-fold decrease in growth as reported by Thangavelu et al. (2018). IAA rapidly oxidizes by plant tissue and higher concentration can cause inhibitory effects on plants. Decrease in plant fresh weight might be due to higher concentration of IAA. Two mechanisms for distribution of IAA in plants can be proposed. (i) IAA was released from CuO NPs in media and taken up by plants independently, (ii) CuO-IAA NPs were taken up by plants and IAA released from CuO NPs inside the plant. It has been reported that generation of reactive oxygen species (ROS) can result in IAA oxidation (Beffa et al. 1990; Huang et al. 2019). Therefore, in the presence of metallic oxide NPs, ROS generation may affect the physiology of IAA which leads to the reduction in biomass. Loach (1987) also reported that for woody ornamental species, higher doses of IAA are harmful, but optimum endogenous concentration of IAA depends on the individual species of plants.

Increase of length, fresh weight, and dry weight of roots at lower concentration of nanoparticles

The root length was prominently increased by application of CuO-IAA NPs as compared to the CuO NPs or IAA (Fig. 8). CuO-IAA NPs significantly increased the root length at 10 and 20 mg/L by 38% and 48%, respectively, as compared to the CuO NPs at the same concentrations. Number of secondary roots formation was higher by the addition of CuO NPs, although increase of NPs concentration decreased the root number. At 5 mg/L CuO-IAA NPs, maximum number of secondary roots was observed. Maximum fresh weight of roots was observed in presence of 5 mg/L CuO-IAA NPs. In presence of NPs, fresh weight was in accordance with the number of roots; higher at lower concentrations and lesser at higher concentrations. Dry weight of the roots was also in parallel with the fresh weight. However, it was more in the presence of CuO NPs as compared to CuO-IAA NPs, although in the presence of both types of NPs, dry weight decreased by an increase in the concentration of NPs. Lower concentration of NPs is considered favorable for plant growth as root length and biomass increased. Higher concentration of NPs reduced the growth of plants. Higher concentration of NPs retards the elongation of roots by blocking the uptake of Mn, Mg, B, Fe, Zn, etc. (Nair and Chung 2014). It has been reported by Khot et al. (2012) and Wu et al. (2012) that NPs affect plant growth, secondary metabolites, physiology, and morphology of plants on the basis of their nature, size, shape, and concentration (Nair and Chung 2014; Deng et al. 2016; Zahir et al. 2019).

Fig. 8.

Fig. 8

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Effect of CuO and CuO-IAA NPs on A root length, and B number of roots of Chickpea plantlets after 19 days of seed germination. Values represent means ± standard errors from triplicates. Small alphabets mentioned on each bar show significant difference among mean values at p < 0.05

Table 1 shows the fresh and dry weight of roots and shoots under CuO and CuO-IAA NPs stress in Chickpea.

Table 1.

Fresh and dry weight of shoots and roots of Chickpea plants under CuO and CuO-IAA NPs stress

Conc. (mg/L) FW (mg/L) DW (mg/L) FW (mg) DW(mg)
Shoots Shoots Roots Roots
Control 398 ± 0.02c 42 ± 0.03 fg 423 ± 01d 50 ± 0.03e
IAA 1 261 ± 0.15g 59 ± 0.08e 407 ± 0.05e 37 ± 0.01f
IAA 2 270 ± 0.17f 78 ± 0.06d 525 ± 0.12b 77 ± 0.03b
CuO 5 633 ± 0.04a 201 ± 0.07a 523 ± 0.08b 99 ± 0.02a
CuO 10 395 ± 0.05c 56 ± 0.02e 521 ± 0.02b 95 ± 0.05a
CuO 20 370 ± 0.05d 46 ± 0.04f 392 ± 0.09f 70 ± 0.04c
CuO 40 360 ± 0.12e 38 ± 0.03g 370 ± 0.11 g 66 ± 0.02c
CuO + IAA 5 413 ± 0.14b 126 ± 0.01b 557 ± 0.03a 77 ± 0.04b
CuO + IAA 10 149 ± 0.04 h 90 ± 0.04c 453 ± 0.12c 60 ± 0.06d
CuO-IAA 20 140 ± 0.02i 79 ± 0.03d 390 ± 0.19f 67 ± 0.03c
CuO-IAA 40 100 ± 0.05j 40 ± 0.02 g 328 ± 0.11 h 50 ± 0.05e
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Values represent means ± standard errors from triplicates

a–jSmall letters mentioned on each value show significant difference among mean values at p < 0.05

Phytochemical screening

Increase of total phenolic and flavonoid contents in the presence of CuO-IAA nanoparticles

Shoots did not show significant change in TPC in all treatments. Presence of IAA in the media exerted stress on plants so increase in TPC was observed. However, stress was also observed at low concentrations of both NPs while with increase in NPs concentration, decrease in TPC was observed. Significant variation in TPC in Chickpea roots was observed when grown in the presence of IAA, CuO NPs, and CuO-IAA NPs. Maximum TPC was observed in CuO NPs treatment, however, treatment with CuO-IAA NPs reduced the stress that resulted in lesser production of phenolic contents in roots (Fig. 9).

Fig. 9.

Fig. 9

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Effects of CuO and CuO-IAA NPs on total phenolics and flavonoid contents in Chickpea plantlets. A TPC in Roots. B TPC in Shoots. C TFC in Roots. D TFC in Shoots. Values represent means ± standard errors from triplicates. Small alphabets mentioned on each bar show significant difference among mean values at p < 0.05 using LSD

The Chickpea shoots showed significant variations in flavonoid contents at all treatments. TFC increased in shoots in presence of IAA, while increase in concentration of CuO NPs decreased the TFC in shoots. However, application of CuO-IAA NPs increased TFC in shoots and by increasing the concentration of NPs. Significant difference in TFC was observed under CuO and CuO-IAA NPs treatments (Fig. 9). These results show that CuO NP-treated plants accumulated more phenolic and flavonoid content than control which exhibits abiotic stress derived elicitation of secondary metabolites (Javed et al. 2022).

As discussed earlier, higher concentrations of NPs negatively affected the morphological characteristics of plantlets which confirm stress on plants. Phytochemicals produced in plants in response to stress. Root and shoot length improved in presence of CuO-IAA NPs which means stress caused by CuO NPs alone was mitigated. Toxic effect resulting from the oxidative state may be allayed by several antioxidative systems such as phytohormones like IAA and polyamines (PAs) that work as vital components of heavy metal stress management (Zafar et al. 2020). IAA is one of the most important signal hormones which functions not only as a plant growth regulator but also as an essential stress tolerance substance (Bashri and Prasad 2016).

Increase in antioxidant activities on increasing concentration of CuO-IAA nanoparticles

Figure 10 shows that CuO NPs produced oxidative response in shoots on increase of concentration of NPs. The same was also observed on application of CuO-IAA NPs. At 5 mg/L, DPPH activity was 50.36% that increased up to 65.18% by increasing the concentration. Concentration-dependent decrease in DPPH activity was observed in roots of plantlets due to CuO-IAA NPs.

Fig. 10.

Fig. 10

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Effects of CuO and CuO-IAA NPs on antioxidant potential of Chickpea plantlets. A DPPH-based free radical scavenging activity in Roots. B DPPH-based free radical scavenging activity in Shoots. C TAC in roots. D TAC in shoots. E TRP in Roots. F TRP in shoots. Values represent means ± standard errors from triplicates. Small alphabets mentioned on each bar show significant difference among mean values at 0.05 probability level using LSD

CuO-IAA NPs increased the TAC activity in shoots, while in case of CuO NPs, TAC increased when concentration of NPs increased up to 20 mg/L and it decreased at 40 mg/L. TAC activity in roots also increased by the presence of IAA as compared to control. Presence of CuO-IAA NPs increased TAC activity in roots and maximum was observed at 40 mg/L.

TRP in control Chickpea shoots was 18.14 µgAAE/mgDW that increased in presence of 1 and 2 mg/L IAA. In the presence of CuO NPs, TRP increased up to 28.90 µgAAE/mgDW at 10 mg/L. However, increase in NPs resulted in decrease of TRP in shoots. TRP in shoots grown at 5 mg/L of CuO-IAA NPs was non-significantly different as compared to control. However, gradual increase in TRP was observed when concentrations of CuO-IAA NPs were increased. Higher TRP response was observed in roots as compared to shoots. In the presence of CuO NPs, TRP was higher in roots at 5 mg/L of NPs that decreased by increase in concentration, however, there was no significant difference in values. In the presence of CuO-IAA NPs, TRP increased by an increase of concentration in the media and maximum was observed at 40 mg/L.

NPs affect plants by the generation of ROS which is the main determinant of oxidative damage (Ma et al. 2015; Saeed et al. 2021). The ROS like O:, O2−, H2O2, OH1− can cause damage to plants (Kumar et al. 2017). However, ROS have significance as signaling molecules and can work in stress response (Ismail et al. 2014). In accordance with these studies, results show that abiotic stress by CuO and CuO-IAA NP-induced antioxidant response and reducing power that increased with increase in the concentration of NPs. This increase of antioxidant activity shows strong detoxification which protects the cell machinery from the toxic behavior of CuO as CuO NPs retard the growth of plants. Radical scavengers (DPPH) can directly quench peroxide radicals which then terminate the chain reaction. Increase of antioxidant activity depicts that there is decrease in the formation of free radicals by inhibiting the activities or expressions of free radical generating enzymes or by enhancing the activities and expressions of other antioxidative enzymes (Lu et al. 2010; Zaeem et al. 2020).

Decrease in antioxidative enzyme activities on increasing concentration of CuO-IAA nanoparticles

It is shown in Fig. 11 that SOD activity was observed 0.22 mM/min/mg FW in roots of control plants. In IAA-treated plants, SOD significantly reduced up to 0.14 mM/min/mg FW at 2 mg/L of IAA. While at 5 mg/L of CuO NPs, SOD increased in roots that decreased by increasing the concentration of NPs. However, when CuO NPs were decorated with IAA, the SOD was only 0.19 that increased by increasing the concentration though there was no significant difference in SOD values in roots grown at 10, 20, and 40 mg/L of NPs. Presence of IAA, CuO NPs, and IAA-CuO-IAA NPs did not cause significant difference in POD values in roots except 1 mg/L of IAA and 10 mg/L of CuO-IAA NPs.

Fig. 11.

Fig. 11

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Effects of CuO and CuO-IAA NPs on antioxidative enzymes of Chickpea roots. A SOD, and B POD production. Values represent means ± standard errors from triplicates. Small alphabets mentioned on each bar show significant difference among mean values at 0.05 probability level using LSD

NPs affect plants by the generation of ROS and these species also behave as signaling molecules. Due to this, it is necessary for plant cells to control the amount of ROS by enzymatic or non-enzymatic antioxidants which includes SOD (Zaka and Abbasi 2017). According to results, increase in concentration of CuO NPs decreased the SOD which indicates that stress was reduced due to the application of CuO NPs attached with surface molecules (IAA). It might be possible that CuO-IAA NPs at lower concentration behave as nanozymes via SOD mimicking for the control of ROS due to their biocompatibility with the plant system (Dhiman et al. 2022; Park et al. 2022). These results are in accordance with the study of Bashri and Prasad (2015) where SOD activity depicted deceasing trend by an increase in IAA concentration. However, it disagrees with the study of Ochoa et al. (2017) that revealed higher catalase (CAT) activity of CuO-IAA NPs with an increasing IAA concentration applied to green pea.

Conclusions and future recommendations

Nanotechnology is an emerging field that has vast applications in agriculture including nanofertilizers for the enhancement of different essential parameters of crop plants. This study demonstrates that CuO NPs affect the growth of Chickpea plants at certain concentrations. Dose-dependent biomass accumulation and enhanced antioxidant activities illustrates that uptake and accumulation is dependent on NPs’ concentration. At 40 mg/L of CuO and CuO-IAA NPs, 75% germination of Chickpea seeds took place. The morphological characteristics of plantlets, i.e., length, fresh weight, and dry weight of shoots and roots increased at lower concentration of CuO and CuO-IAA NPs and vice versa. Similarly, antioxidant enzymes activities also decreased at higher concentration of NPs. However, enhancement of phytochemicals (TPC and TFC) and non-enzymatic antioxidant activities was revealed at higher concentrations of NPs. The CuO-IAA NPs declined antioxidants production (TAC, TRP, %DPPH-FRSA) by decreasing the oxidative stress produced by CuO NPs due to the presence of IAA functioning as stress inhibitor besides phytohormone.

The rationale of this study was that Cu plays important role in the growth of plants, hence exposure of different plants to CuO NPs at appropriate concentrations could open new avenues of research in the nanobiotechnology field. The 30.4 nm-sized CuO-IAA NPs designed have proven potential candidates to be used as nanocarriers in plants at lower concentrations. They also have ability to reduce the toxicity caused by higher concentrations of NPs. It can also be concluded from phytochemical assays that higher concentrations of CuO-IAA NPs behave as abiotic stress and is useful tool to enhance the production of secondary metabolites (antioxidants) in plants.

In future, BET analysis could be performed to better illustrate the surface area of nanocarrier utilized in the study. The experiments should be planned to find the influence of other elements like Mn, Mg, B, Fe, and Zn on the Chickpea plantlets. In addition, the combined effect of nutrients and microbes could be studied using soil as culture medium. Moreover, histological fingerprinting and transcriptional expression of genes encoding metal transporter in NPs-treated roots should be conducted using real-time PCR.

Acknowledgements

Authors are thankful to the Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan for providing all research facilities.

Author contributions

SH: writing—original draft, formal analysis, data curation, validation RJ: investigation, validation, writing—original draft, writing—review and editing, visualization AK: writing—original draft, data curation AS: writing—original draft MZ: conceptualization, methodology, resources, supervision, project administration.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Research involving human participants and/or animals

Our research don’t involve humans or animals.

Informed consent

Not Applicable.

Contributor Information

Rabia Javed, Email: rjaved@grenfell.mun.ca.

Muhammad Zia, Email: ziachaudhary@gmail.com.

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