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Article

Brassinosteroids Alleviate Ethylene-Induced Copper Oxide Nanoparticle Toxicity and Ultrastructural and Stomatal Damage in Rice Seedlings

by
Wardah Azhar
,
Abdul Salam
,
Ali Raza Khan
,
Irshan Ahmad
and
Yinbo Gan
*
Zhejiang Key Lab of Crop Germplasm, Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(8), 907; https://doi.org/10.3390/agriculture15080907
Submission received: 7 February 2025 / Revised: 13 April 2025 / Accepted: 17 April 2025 / Published: 21 April 2025
(This article belongs to the Section Crop Production)
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Abstract

:
Nanoparticle contamination has been associated with adverse impacts on crop productivity. Thus, effective approaches are necessary to ameliorate NP-induced phytotoxicity. The present study aimed to investigate the efficacy of brassinosteroids and ethylene in regulating CuO NPs toxicity in rice seedlings. Therefore, we comprehensively evaluated the crosstalk of 24-Epibrassinolide and ethylene in regulating CuO NP-induced phytotoxicity at the physiological, cellular ultrastructural, and biochemical levels. The results of the study illustrated that exposure to CuO NPs at 450 mg/L displayed a significant decline in growth attributes and induced toxic effects in rice seedlings. Furthermore, the exogenous application of ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) at 20 µM with 450 mg/L of CuO NPs significantly enhanced the reactive oxygen species (ROS) accumulation that led to the stimulation of ultrastructural and stomatal damage and reduced antioxidant enzyme activities (CAT and APX) in rice tissues. On the contrary, it was noticed that 24-Epibrassinolide (BR) at 0.01 µM improved plant biomass and growth, restored cellular ultrastructure, and enhanced antioxidant enzyme activities (CAT and APX) under exposure to 450 mg/L of CuO NPs. In addition, brassinosteroids reduced ROS accumulation and the toxic effects of 450 mg/L of CuO NPs on guard cells and the stomatal aperture of rice seedlings. Interestingly, when 0.01 µM of brassinosteroids, 20 µM of ACC, and 450 mg/L of CuO NPs were applied together, BRs and ethylene showed antagonistic crosstalk under CuO NP stress via partially reducing the ethylene-induced CuO NP toxicity on plant growth, cellular ultrastructure, stomatal aperture, and guard cell and antioxidant enzyme activities (CAT and APX) in rice seedlings. BR supplementation with ACC and CuO NPs notably diminished ACC-induced CuO NPs’ toxic effects on all of the mentioned attributes in rice seedlings. This study uncovered the interesting crosstalk of two main phytohormones under CuO NPs stress, providing basic knowledge to improve crop yield and productivity in CuO NPs-contaminated areas.

1. Introduction

Recently, nanotechnology has emerged as one of the most promising technologies because of its applications in various sectors [1,2]. Nanoparticles are small atoms or molecules with at least one dimension less than 100 nm. They can be either naturally occurring or are produced synthetically, and exhibit a high surface area to volume ratio [3,4]. These distinctive properties enhance the binding capacity and dispersibility of nanoparticles in the solution, resulting in higher reactivity compared to the bulk particles [5].
Metal nanoparticles represent a novel category of contaminants, exhibiting the characteristics of both metals and nanoparticles (NPs). These unusual materials have elicited worldwide concern regarding potential environmental toxicity related to their structure and distinctive chemical and physical properties compared to bulk metals [6].
Copper oxide nanoparticles (CuO NPs) are one of the most important engineered NPs, having enormous usage in various fields including environment, agriculture, electronics, construction, etc. [7,8,9,10,11]. In addition, CuO-NPs possess various applications in coatings, healthcare, polymers, and agriculture, including fungicides, nano insecticides, and wood preservatives. Nonetheless, their rising application in agro-ecological systems has drawn scientific attention because of their potential toxicity [12,13,14,15]. It is estimated that the world may consume about 200,000–830,000 kg of Cu-based NPs every year from 2020 to 2025 (global market). Around 200 tons of nano copper were produced at the start of the previous decade, with estimates indicating an increase to 1600 tons by 2025 [16,17,18]. Approximately 11 metric tons were released into the water and 36 metric tons were deposited in the soil [19,20]. Approximately 95% of the total industrial CuO NP residue is known to finally accumulate in the soil [16,21]. Copper oxide nanoparticles are regarded as safe nutrients; nonetheless, numerous investigations have demonstrated their toxicity due to the induction of oxidative stress at elevated dosages [22].
Based on the above exposure of CuO NPs to the environment, they may likely induce high CuO NPs concentrations in soil. Their huge level of applications and their high demand increase their release into the environment [23]. In addition, their excessive usage may negatively affect plants, owing to their tendency to accumulate in soil and their water solubility [24], thus enhancing their bioaccumulation in different crops [25,26].
Plants are primary producers and fundamental trophic levels in the food chain of terrestrial ecosystems for living organisms [27]. Different studies have shown that CuO NPs induced toxic effects in maize [18], and Cucumis melo [28]. Their excessive bioaccumulation in food crops may cause serious toxic effects on human health [29,30]. CuO NPs have been shown to induce toxicity in various plant species [18,28,31]. CuO NPs induced modulation of antioxidative defense and photosynthetic performance of Syrian barley [32]. It has also been illustrated that CuO NPs accelerate ROS accumulation in plants [18], indicating the unfavorable impact of copper-based NPs on edible plants. To cope with these oxidative damages, plants improve their antioxidant defense system, which is important in scavenging ROS accumulation [33,34]. This excessive production of ROS stimulates the antioxidant defense system, including ascorbate peroxidase (APX) and catalase (CAT) [35,36]. The CAT and APX are very important parts of the antioxidant defense system and actively convert H2O2 into H2O and O2 [37].
The improper disposal of CuO NPs may create environmental contamination. Consequently, a thorough investigation of the potential hazards associated with using CuO NPs for plant production is essential. Therefore, overcoming the adverse effects of CuO NPs from edible crops is necessary to protect living organisms from their toxic effects. It is crucial to solve the safety issues regarding metallic NPs, especially Cu-based NPs, due to their toxic impacts on the environment and agriculture sectors [31,38,39]. In this view, we investigated the role of different phytohormones (brassinosteroids and ethylene) in regulating CuO NPs toxicity in rice seedlings. Preventing CuO NPs toxicity in rice plants would be an important step in reducing the environmental impacts of CuO NPs pollution.
Considering the various functions of phytohormones in mediating abiotic stress resistance and activating systematic adaptive mechanisms, this study aimed to explore the role of BRs and ethylene in the regulation of CuO NPs-induced toxicity in rice seedlings. Phytohormones not only regulate plant growth and development, but also play an important role in plant tolerance mechanisms against environmental stresses [40]. Brassinosteroids are known as a class of plant polyhydroxy steroids that engage in plant growth and developmental processes [41]. In addition, BRs help plants to regulate biotic and abiotic stress factors [42,43,44]. The exogenous application of BRs improved photosynthesis and chlorophyll content in cucumber plants [45]. Recently, it has been reported that exogenous EBR can increase the Fv/Fm and chlorophyll content and reduce the malondialdehyde (MDA) content under Cd stress in Solanum nigrum L. seedlings [46]. It also ameliorated the Cd toxicity in Raphanus sativus seedlings by improving the antioxidant defense system [47]. However, the potential mechanisms of BRs to diminish CuO NP-induced oxidative stress in rice seedlings are not yet known. To date, how brassinosteroids interact with ethylene and mitigate CuO NP-induced phytotoxicity at the physiological, biochemical, and cellular ultrastructural levels in rice seedlings has not been investigated.
Ethylene is a simple gaseous phytohormone that has the main function in various physiological processes [48], such as ethylene-induced oxidative damage and reduced plant growth under different abiotic stresses [49,50]. Considering the involvement of ethylene in biotic and abiotic stresses, we expect that ethylene biosynthesis precursor (ACC) may accelerate CuO NPs-induced oxidative damage in rice seedlings. Therefore, the efficacy of BRs in interacting with ethylene to mitigate the ethylene-induced toxic effects of CuO NPs from rice crops is crucial to avoid the emerging contamination of CuO NPs in the food chain.
BRs influence ethylene biosynthesis by regulating the activities of ACS and ACO [51]. There are two possible scenarios for crosstalk between BRs and ethylene: BRs may regulate the transcriptional or post-transcriptional levels of ethylene. Previously, it has been reported that seed treatments with exogenous BRs exhibited an increase in ACS5 protein stability by changing its half-life, thus displaying elevated ethylene levels [51]. BRs regulated the ethylene biosynthesis in a dose-dependent manner, so it may have acted as a positive or negative regulator, based on the exogenous supply [52]. The crosstalk between different phytohormones occurs at the stage of their biosynthesis, gene expression, or signal transduction [53]. Recently, it was proposed that BRs mediate stress tolerance through crosstalk with other phytohormones [54]. The synergistic or antagonistic crosstalk of phytohormones is a broad and complex topic and attracts the attention of scientists. It has been found through investigations that ethylene and BRs altered ascorbic acid–glutathione (AA-GSH) levels in tomato plants. BRs and ethylene were shown in the study to exhibit antagonistic responses in the regulation of ascorbic acid content (AA) in tomatoes. BRs enhanced the accumulation of AA content. On the contrary, ethylene reduced the AA accumulation [55]. To date, the crosstalk (antagonistic or synergistic) between BRs and ethylene under CuO NPs stress in rice crops has not been investigated.
Rice is one of the most important staple foods in Asia [56]. Various abiotic stresses have been noted to cause a decline in rice growth and development, reducing productivity [57,58]. Considering the nutritional importance and harsh environmental threat to rice crops, there is an immediate need to mitigate the toxic effects of the emerging contamination of CuO NPs on this staple food. Therefore, the present study aimed to explore the role of two important phytohormones (brassinosteroids and ethylene) in regulating CuO NP toxicity in rice seedlings. It was hypothesized that the exogenous application of BRs may ameliorate the toxic effects of CuO NPs at physiological, biochemical, and cellular ultrastructural levels in rice seedlings. The credible evidence presented in this study also confirms that ethylene (ACC) may further accelerate CuO NP-induced toxicity and oxidative damage to these attributes. In addition, exogenous BRs may show antagonistic crosstalk with ethylene and reverse ethylene-induced CuO NPs toxic effects on rice seedlings. The possible antagonistic relationship between BRs and ethylene in the amelioration of CuO NP toxicity was explored in rice seedlings.

2. Materials and Methods

2.1. Characterization of CuO NPs

The morphology and size of CuO NPs has been detailed in a previous study [59]. For the scanning electron microscopy (SEM) analysis, using carbon double-sided glue tabs, the samples were fixed on an aluminum stub and enclosed by conductive palladium film. Scanning electron microscopy (SEM) (TM-1000, Hitachi, Japan) was used to observe the sample. In addition, the elemental composition of the sample was determined by a field emission scanning electron microscope, which was linked with energy dispersive X-ray (FESEM-EDX).

2.2. Plant Material and Growth Conditions

Rice (cv. Nipponbare) seeds were obtained from the College of Agriculture and Biotechnology, Zhejiang University, China. Seeds were surface sterilized with 75% ethanol for 1 min, followed by 10% sodium hypochlorite for 20 min. Distilled water was used to wash the seeds five times [60,61]. Sterilized seeds were placed in Petri dishes covered with Whatman No. 1 filter paper and moistened with 10 mL distilled water. Petri dishes containing seeds were placed in the dark at 30 °C for 3 days. After that, the seeds with uniform germination were transferred to Yoshida nutrient solution [62] for 7 days. The seedlings were placed in a growth room with a 14/10 h (light/dark) photoperiod (600 µmol m−2 s−1 intensity) with relative humidity (65–70%) and a temperature of 30 °C and 28 °C for the day/night, respectively, as we described [63]. The plants were subjected to different treatments for a further 7 days.

2.3. Determination of Biomass Accumulation

To find out the toxic impacts of CuO NPs on biomass accumulation, the seedlings were exposed to different treatments of CuO NPs (0, 450 mg/L), ACC-CuO NPs (20 µM + 450 mg/L), BR-CuO NPs (0.01 µM + 450 mg/L), and BR-ACC-CuO NPs (0.01 µM + 20 µM + 450 mg/L) in nutrient solution. After 7 days of treatment, the plants were harvested, washed with tap water, and cleaned again with distilled water. The root and shoot were separated and dried with a paper towel. A digital electric balance was used to measure the seedlings’ fresh weight (FW). The samples were first dried at 85 °C for 15 min, then at 65 °C until fully dried, after which the dry weight (DW) was measured.

2.4. Photosynthetic Pigment Measurements

Chlorophyll (Chl) a, and Chl b were measured using a spectrophotometer (model UV-2600) [64]. Fresh rice seedlings’ leaves were weighted and incubated in 85% acetone. The chlorophyll contents (a, b) were determined at a wavelength of 452.5 and 644 nm spectrophotometrically [65] and calculated as described by [66].

2.5. Measurements of Lipid Peroxidation

Malondialdehyde content in the roots and shoots was analyzed using the protocol described by [67]. To investigate the toxic effects of CuO NPs on cell membrane lipid peroxidation, 0.2 g shoot and root samples were weighted and homogenized in (0.67%) 2-thiobarbituric acid (TBA), which was made in 10% trichloroacetic acid (TCA). The homogenized samples were incubated at 95 °C for 30 min and cooled in an ice bath. A microplate reader (Bio-Rad, Hercules, CA, USA) was used to measure the MDA contents at a wavelength of 532 and 600 nm [59].

2.6. NBT and DAB Staining

After 7 days, the seedlings were treated with different concentrations of CuO NPs (0, 450 mg/L), ACC-CuO NPs (20 µM + 450 mg/L), BR-CuO NPs (0.01 µM + 450 mg/L), and BR-ACC-CuO NPs (0.01 µM + 20 µM + 450 mg/L). The ROS accumulation was measured by nitroblue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) staining assays. The chlorophyll from the shoots was removed by dipping the samples in absolute ethanol and heating them in a water bath. The samples were stained according to the protocol described previously [18]. The stained samples were washed and mounted on the glass slides. The Leica stereomicroscope (DMIRB Leica MZ 95, Munich, Germany) was used to take images of the stained leaves, as described previously [50,59].

2.7. Scanning Electron Microscopy (SEM)

To observe the stomatal shape and assess guard cell damage in the differently treated samples, leaf segments without veins were carefully excised from the seedlings exposed to CuO NPs (0, 450 mg/L), ACC-CuO NPs (20 µM + 450 mg/L), BR-CuO NPs (0.01 µM + 450 mg/L), and BR-ACC-CuO NPs (0.01 μM + 20 μM + 450 mg/L) for 7 days. Instantly, the samples were dipped in glutaraldehyde (2.5%) in phosphate buffer (0.1 M) at pH 7. Then, for 1–2 h, the samples were post-fixed with osmium tetroxide (1.0%) in PBS. After that, the specimens were washed 3 times in phosphate buffer (0.1 M at pH 7.0). Furthermore, the samples were washed with different concentrations (30%, 50%, 70%, 80%, 90%, 95%, and 100%) of ethanol. The specimens were eventually dried out by using liquid carbon dioxide and examined under a scanning electron microscope (Hitachi Model SU-8010, Hitachi, Japan) by following the protocol described by [18,33,65].

2.8. Transmission Electron Microscopy (TEM)

The leaves were cut into small pieces, excluding the veins, from the seedlings exposed to CuO NPs (0, 450 mg/L), ACC-CuO NPs (20 µM + 450 mg/L), BR-CuO NPs (0.01 µM + 450 mg/L), and BR-ACC-CuO NPs (0.01 µM + 450 mg/L). The samples were dipped in glutaraldehyde (2.5%) in phosphate buffer (0.1 M) at pH 7.0. Then, the specimens were washed three times with PBS and post-fixed for 1–2 h with osmium tetroxide (1.0%) in phosphate buffer, followed by washing three times with phosphate buffer. The samples were dehydrated each for 15 min by using a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%). Further, they were submerged in Spurr’s resin overnight for 9 h heated at 70 °C. For transmission electron microscopy (TEM) analysis, the small pieces of specimens were placed on copper grids and observed under TEM (Hitachi H-7650) following the protocol described by [18,33,65].

2.9. Measurement of Antioxidant Enzyme Activities

To investigate the antioxidant enzyme activities, 0.3 g samples of the root and shoot were homogenized in (50 Mm) phosphate buffer (pH 7.8) in a pre-cooled pestle and mortar. After homogenization, the samples underwent centrifugation (12,000 rpm) for 15 min at 4 °C. The resulting supernatant was used to measure the following antioxidant enzyme activities using a spectrophotometer (Shimadzu, Kyoto, Japan):
The catalase (CAT) activity was determined at 240 nm for 30 s using a spectrophotometer. The reaction solution comprised phosphate buffer (50 mM, pH 7.8), enzyme extract, and (300 mM) H2O2. The activity was determined via following the method of [18].
The ascorbate peroxidase (APX) activity was determined via following the protocol previously described by [68]. The reaction solution contained (7.5 mM) ascorbic acid, enzyme extract, (50 mM) phosphate buffer (pH 7.8) and (300 mM) H2O2. The activity was recorded at a wavelength of 290 nm for 60 s using spectrophotometer.

2.10. Statistical Analysis

The experiments were performed in three replicates. The data were analyzed with one-way ANOVA by using statistics software (Version 8). The significance difference was determined by using the least significant difference (LSD) at the probability level (p < 0.05). The different lowercase letters showed the significant difference between the treatments, as we described previously [65]. The error bars showed the standard error (S.E) in the figures.

3. Results

3.1. Characterization of CuO NPs

The scanning electron microscopy results demonstrated that the CuO NPs had uniform size (Figure 1A). The elemental composition and EDX spectra of the CuO NPs are presented in (Figure 1B,C). The EDX spectra exhibited peaks corresponding to copper (Cu) and oxygen (O), confirming the synthesized nanoparticles’ purity. Additionally, we conducted EDX mapping to validate the findings of EDX spectroscopy. The results indicated that the sample predominantly comprised copper and oxygen, exhibiting strong signals (Figure 1D–G). These results collectively demonstrated the high purity of the CuO NPs used in this study.

3.2. BRs Reverse Ethylene-Induced CuO NP Toxicity and Improves Plant Growth Attributes

As depicted in Figure 2A, CuO NPs stress at higher concentrations (450 mg/L) induced the clear inhibition of root and shoot growth in rice seedlings. CuO NPs stress significantly hampered seedling height. The exogenous application of the ethylene biosynthesis precursor (ACC) further inhibited the growth of rice seedlings under CuO NPs stress. In contrast, exogenous brassinosteroids (BRs) significantly alleviated the growth inhibition induced by CuO NPs. In addition, plant phenotype was improved by the application of ACC in combination with BRs under CuO NP stress. All these above chemical treatments suggested that BRs not only mitigated the CuO NPs-induced plant growth inhibition but also reversed the ethylene-induced negative effects on rice growth under CuO NPs stress (Figure 2A). Biometric parameters were employed to assess the responses of different phytohormones (ethylene and BRs) under CuO NPs stress in rice seedlings. The exogenous supply of CuO NPs significantly reduced the fresh weight (FW) (50.55%) as compared to the control (Figure 2B). The co-application of CuO NPs and ACC induced significant negative effects on plant growth and further reduced the FW (56.04) as compared to the CuO NP stress alone. In addition, BRs reduced these deleterious effects of CuO NPs and a 13.19% reduction in FW was recorded under BR + CuO NPs treatments compared to the relative controls. Furthermore, a clear improvement in root and shoot lengths was also observed (Figure 2C,D). Interestingly, the reduced biomass and plant growth under ACC treatments were partially recovered when ACC was applied in combination with BRs and CuO NPs (Figure 2A–D). Previously, it was found that BRs with lower concentrations (10 nM) negatively regulated ethylene biosynthesis and improved root growth in Arabidopsis thaliana [52]. Collectively, these results suggested that BRs actively mitigate the adverse effects of CuO NPs and ethylene on rice growth and biomass under CuO NPs stress.

3.3. BRs Inhibit Ethylene-Induced Chlorophyll Degradation in Rice Seedlings Under CuO NPs

Our results revealed that CuO NPs significantly reduced the chlorophyll content. The combined application of ACC with CuO NPs induced severe toxic effects on the chlorophyll and further degraded the chlorophyll content (Figure 3A,B). However, the application of BRs in nutrient media in the presence of CuO NPs significantly reversed the CuO NPs-induced chlorophyll reduction in rice seedlings. Furthermore, BRs, in the presence of ACC and CuO NPs, actively reduced the ethylene-induced chlorophyll reduction (Figure 3A,B). Overall, these results suggested that exogenous BRs not only improved the chlorophyll content accumulation but also significantly reversed the negative effects of ethylene on chlorophyll pigments in the leaves of rice seedlings under CuO NP stress.

3.4. BRs Minimize Ethylene-Induced Oxidative Damage in Rice Seedlings

To evaluate the potential mechanisms of BRs in eliminating the ethylene-induced ROS accumulation under CuO NPs, we observed H2O2 and O2•− accumulation and their verifications via DAB and NBT staining assays. Histochemical staining analyses revealed that the supply of CuO NPs alone had significantly induced ROS accumulation in shoots, as evidenced by the dark brown and dark blue colors. With the addition of ACC into the nutrient solution along with the CuO NPs, the production of H2O2 and O2•− was further increased as compared to CuO NPs treatments alone (Figure 4A–C). Meanwhile, BR exposure in combination with CuO NPs minimized the ROS production, indicating that BRs suppressed the accumulation of H2O2 and O2•− content in plant tissues. Interestingly, simultaneous treatments with ACC and BRs under CuO NPs noticeably reduced the ROS accumulation. These results suggested that BRs significantly reduced the ethylene-induced ROS accumulation under CuO NPs stress.

3.5. BRs Reverse the Ethylene-Induced MDA Content Accumulation

Rice seedlings were exposed to CuO NPs (0, 450 mg/L), ACC-CuO NPs (20 µM + 450 mg/L), BR-CuO NPs (0.01 µM + 450 mg/L), and ACC-BR-CuO NPs (20 µM + 0.01 uM + 450 mg/L) for 7 days. As compared to the control, CuO NPs (450 mg/L) significantly induced MDA accumulation. The combined treatments of ACC and CuO NPs further enhanced the MDA accumulation. This suggested the involvement of ethylene in lipid membrane peroxidation under CuO NPs stress. Followed by the H2O2 and O2•− accumulation, MDA content significantly declined upon the co-exposure of BRs and CuO NPs. Moreover, our results showed that BRs significantly restricted MDA accumulation by reducing the ROS production in both roots and shoots. Interestingly, while the application of ACC alone increased MDA levels, this increase was mitigated when ACC was co-applied with BRs under CuO NPs stress (Figure 4D,E). These results further confirm that BRs alleviate the ethylene-induced CuO NPs toxicity, as shown by the lower levels of oxidative markers.

3.6. BRs Ameliorate Ethylene-Induced Stomatal Damage in Rice Seedings

The results of scanning electron microscopy disclosed that CuO NPs stress damaged the stomatal aperture. The guard cells and stomatal aperture of the seedlings subjected to ACC-CuO NP stress were more damaged as compared to CuO NPs stress alone (Figure 5B,G). Furthermore, ACC-CuO NPs treatment exacerbated damage to the stomatal aperture and guard cells. Notably, the stomata of leaves exposed to BR-CuO NPs were significantly wider and longer compared to those subjected to CuO NPs stress alone (Figure 5D,I). Considering that BRs facilitate the osmotic retention of water in guard cells, leading to stomatal opening, its application may play a crucial role in mitigating CuO NPs-induced stomatal closure. It was observed that BRs reversed the ethylene (ACC)-induced stomatal damage under CuO NPs stress (Figure 5E,J). Collectively, these results suggest that BRs may overcome the toxic effects of ethylene-induced stomatal damage in rice seedlings.

3.7. BRs Reduce Ethylene-Induced Ultrastructural Damages Under CuO NP Stress

The transmission electron microscopy (TEM) analysis of the mesophyll cells of leaves revealed clear nuclear membranes with a round nucleus, well-organized thylakoid membranes, and well-shaped chloroplast structures under control conditions (Figure 6A). In contrast, those treated with 450 mg/L of CuO NPs displayed scattered and irregular shapes along with the nucleus, abnormal chloroplast, disruptive thylakoids membranes, broken cell walls, and cell membranes (Figure 6B). TEM analysis of ACC-CuO NPs-treated samples displayed the disastrous shape of the cells (Figure 6C). Furthermore, it was noticed that ACC treatments drastically damaged the nucleus shape, resulting in a nucleus without the nucleolus, swollen mitochondria with disturbed cristae, broken cell wall, and dissolved thylakoid membranes with abnormal chloroplast shapes (Figure 6C). Dissimilarly, BRs mitigated the oxidative damages and reversed the negative effects of CuO NPs on cell organelles. The TEM observation of BR-CuO NP-treated seedlings exhibited a round-shaped nucleus with the nucleolus, clear mitochondria with normal cristae, and chloroplasts with well-arranged thylakoid membranes (Figure 6D). Interestingly, the co-exposure of BRs, ACC, and CuO NPs demonstrated destructive cell organelles, but the BRs effectively recovered the negative effects of ACC on the ultrastructure of mesophyll cells under CuO NPs (Figure 6E). Collectively, these results showed that BRs significantly mitigate the ethylene-induced toxic effects of CuO NPs on the ultrastructure of rice seedlings.

3.8. BRs Improve the Surface of Rice Root Exposed to CuO NPs

To assess and verify the toxic impacts of CuO NPs on the root morphology (texture), we employed scanning electron microscopy (SEM). The application of CuO NPs alone suppressed the root growth and altered the root surface (Figure 7B). No such disparity was detected in the root texture under the control and BR-treated plants in (Figure 7A) and (Figure 7D), respectively. On the contrary, rice seedlings treated with ACC in the presence of CuO NPs showed intensive damage to the root texture as compared to applications of CuO NPs alone (Figure 7C). Notably, the combined treatment of ACC and BRs clearly reversed the ACC-induced root texture damages (Figure 7E). This indicates that BRs not only reversed the toxic effects of CuO NPs but also ameliorated ethylene-induced CuO NPs toxicity on the root texture and morphology.

3.9. BRs Reversed Ethylene-Reduced Antioxidant Enzyme Activities Under CuO NPs

The results showed that CuO NPs significantly reduced the CAT and APX activities in both the root and shoot tissues of rice seedlings (Figure 8A–D). The precursor of ethylene biosynthesis (ACC) further reduced these enzyme activities under CuO NPs stress. However, the co-application of BRs and CuO NPs increased the antioxidant enzyme activities compared to the exposure of rice seedlings to CuO NPs alone (Figure 8A–D). Interestingly, when ACC was supplied in combination with BRs under CuO NP stress, BRs reversed the ethylene-induced toxic effect on antioxidant enzyme activities, and these activities were found to be higher than in seedlings treated with CuO NPs alone (Figure 8A–D). These results demonstrated that BRs actively reversed ethylene’s negative impacts on rice seedlings’ antioxidant defense system under CuO NPs stress.

4. Discussion

Few studies have reported the toxic effects of CuO NPs on different plant species, such as pakchoi (Brassica campestris L.) [25], Cucumis melo [28], and Arabidopsis thaliana [59]. However, the potential mechanisms of brassinosteroids (BRs), adapted to reverse the ethylene-induced CuO NPs toxicity in different plants, have not been investigated. This report provided promising results that supported the important function of BRs in mitigating ethylene-induced CuO NPs toxicity in rice seedlings.
Our results showed that excessive CuO NPs concentrations significantly reduced plant growth and biomass accumulation (Figure 2A–D). The CuO NP-induced growth inhibition may have been due to the root damage (Figure 7). Rice seedlings subjected to ACC in conjunction with CuO NPs had severe root texture impairment relative to those exposed to CuO NPs alone. Moreover, BRs alleviated the detrimental impacts of ethylene-induced CuO NPs toxicity on root texture damage, indicating that the augmented root length and biomass of rice seedlings may be attributed to BRs’ role in enhancing root texture under the simultaneous stress of ethylene and CuO NPs (Figure 7). The seedlings treated with BRs alone had higher root and shoot lengths than other treatments, indicating a positive response to BRs that helped to reduce the CuO NPs-induced toxicity on these growth traits. These outcomes aligned with the result reported by [69], who noticed that EBL improved the seedlings’ growth in mung bean plants under zinc stress. Furthermore, an instance of this can be observed in the report conducted by [70], where they observed that treatment with EBL had an effective positive impact on plant growth under chromium stress in tomato plants. Recently, a study reported that foliar spray with a brassinosteroid analog (DI-31) effectively enhanced drought tolerance via improving photosynthetic efficiency and plant growth in Solanum quitoense Lam plants [71]. Furthermore, in the combined treatment with BRs and ethylene, BRs significantly reversed the ethylene-mediated reduction in plant growth and biomass accumulation (Figure 2A–D). The pre-treatment of lettuce plants with the BR analog (D-31) evoked ethylene biosynthesis and significantly improved the salinity tolerance [54].
Reactive oxygen species play a dual role in plant physiology; they function as signal molecules, and on the other hand, they induce toxicity and oxidize the components of cells [72]. In this work, CuO NPs treatment considerably raised H2O2 content and O2− accumulation in rice leaves, which was verified by DAB and NBT staining assays (Figure 4A), indicating the presence of oxidative stress. ROS are very active, and excessive ROS formation causes membrane lipid peroxidation, which leads to a rise in MDA content accumulation [72]. As shown in our figure (Figure 4D,E), MDA levels were also elevated, showing that ROS may cause cell oxidative damage. Similar results were obtained by Khan et al. (2023), who observed that CuO NPs stress generated high levels of ROS and caused MDA content accumulation in maize plants [18]. In contrast to the deleterious effects of CuO NPs, exogenous BR application considerably reduced H2O2, O2−, and MDA levels in rice leaves under CuO NPs stress. These results were consistent with prior research, where they found that EBR significantly lowered H2O2, O2−, and MDA levels in Cu-stressed grape leaves and roots [73]. Soares et al. (2016) observed that applying 24-EBL reduced H2O2, O2−, and MDA levels in Solanum nigrum plants exposed to Ni metal [74]. The mechanisms by which BR application reduced the H2O2, O2−, and MDA induced by ethylene and CuO NPs may have involved the activation of antioxidant enzyme systems [75].
These stress indicators, H2O2, O2−, and MDA, were further enhanced in plants exposed to the ethylene biosynthesis precursor (ACC) with CuO NP stress. Similarly, previous reports showed a similar effect of ethylene in Arabidopsis thaliana leaves under nanoparticle exposure [59]. More importantly, the exogenous application of BRs reversed the ethylene-induced ROS accumulation under CuO NPs stress. These observations suggested that BRs might have inhibited ethylene biosynthesis and ameliorated the ethylene-induced oxidative damage under CuO NPs in rice seedlings. BRs were also found to reduce ethylene biosynthesis and significantly reversed the adverse effects of ethylene on ascorbic acid (AA) (which improved the antioxidant defense system) in tomato plants [55].
The impact of different NPs on plant anatomy has been investigated through various microscopic techniques, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM) [76]. The SEM observation of the root surfaces displayed damage on the root surface under CuO NPs stress (Figure 7B). Our results aligned with previous findings, where CuO NPs were observed to cause external damage to rice roots, as evidenced by SEM analysis [77]. They reported the external morphological changes in the roots of rice plants under CuO NPs exposure. The changes in root morphology under all treated seedlings indicated the ability of rice plants to adapt to external environmental stress. The changes in the root morphology under CuO NP exposure might have been attributed to the absorption of NPs on the root surface, which led to the reduction in trans-root potential, which is very important for the uptake of water-containing essential ions [78]. Nanoparticles adhere, contact the cell wall surfaces, and block the movement of nutrients between the cell and the surrounding medium [79]. Our results showed that CuO NPs in combination with ethylene (ACC) reduced the root length (Figure 2C), as ethylene had negative effects on cell expansion and restricted the roots [80]. Furthermore, it induced irregulation in the root surface of the meristematic zone (Figure 7C). These adverse changes in the root morphology via ACC application led to a reduction in water uptake and ions essential for plant growth and development [78]. On the contrary, BRs minimized the adverse effect of CuO NPs on root growth (Figure 2) and their surface morphology (Figure 7D). Plants can express a rapid response under BR applications by increasing root and shoot growth [81]. Our results align with a previous study, which reported that overexpressing the BR receptor (BR1) significantly improved the size of mesophyll and epidermal cells [82].
Our findings are also aligned with previous studies on the toxicity of CuO NPs in wheat seedlings [83], where they demonstrated that rice husk-derived biochar mitigated the adverse effects of CuO NPs on wheat growth, proposing that biochar forms a protective barrier on the root surface, preventing the uptake of CuO NPs into plant cells and thereby reducing their biotoxicity. Similarly, our study observed reduced damage to the root surface of rice seedlings treated with brassinosteroids (BRs) under CuO NP stress. This suggests that BRs may inhibit the contact or entry of CuO NPs into rice roots. Furthermore, the lesser damage to the leaf cellular ultrastructure in BR-treated seedlings further supports the idea that BR exposure reduces CuO NPs-induced biotoxicity.
Brassinosteroids might have been involved in the elongation of mesophyll and epidermal cells and the improvement of rice growth under CuO NPs stress (Figure 2A). Brassinosteroids improved ethylene synthesis through the stabilization of ACS5 and ACS9 proteins [51]. These responses were also detected in other crops, such as mung bean and Zea mays [84,85]. These antagonistic and synergistic responses indicate the complex interaction of BRs with ethylene. However, in our study, 0.01 µM of exogenous BRs reversed ethylene-induced root surface damage in rice seedlings (Figure 7E); one report also highlighted the dose-dependent effect of brassinosteroids (BRs) on ethylene synthesis and their subsequent impact on root growth, suggesting an antagonistic relationship between BRs and ethylene under CuO NPs stress [52].
Different studies have mentioned the toxic effects of NPs on plant cell organelles [59,86]. The results of the TEM observation clearly showed the changes in the ultrastructure of the leaves (Figure 6). Disorganized thylakoid membranes, abnormal plastoglobuli, destructed nucleus with the nuclear membrane, disturbance and swelling in the mitochondria, and destruction in the mitochondrial cristae were found under CuO NPs stress (Figure 6B). Similarly, CuO NPs distorted thylakoid membranes in rice seedlings [77]. It was observed that excessive levels of CuO NPs induced distortion in the cell organelles of maize [18] and barley plants [87]. In addition, the ethylene biosynthesis precursor (ACC) further damaged these cell organelles with CuO NPs (Figure 6C). This aligned with the negative role of ethylene in cellular ultrastructure damage in plants under different stresses [49,59,86]. Strikingly, we noticed that exogenous BRs with CuO NP stress minimized the ultrastructural damages by reducing the stress indicators (Figure 4A–E). The extent of damage was found to be lower in BR-treated seedlings in the presence of CuO NPs (Figure 6D). Studies have shown that exogenous BRs stabilized the thylakoid membranes and chloroplast structures under waterlogging conditions in maize seedlings [88]. A recent study illustrated that BRs restored the cellular ultrastructural damages in Brassica napus under parasitic weed stress [89].
Interestingly, BRs also mitigated the toxic effects of ACC on cell organelle damage under CuO NPs stress (Figure 6E). ROS generation at a higher rate also caused sub-cellular damage [90]. Similarly, exogenous ACC application accelerated ROS generation and oxidative damage in rice tissues, which might have been responsible for ultrastructural damage (Figure 4A–E). The lower extent of damage in BR-treated samples and with BR-ACC application evidenced that BRs reversed ethylene-induced toxic effects on the cell organelles of rice seedlings under CuO NPs stress.
Similarly, the SEM results of the leaves’ epidermis showed that CuO NPs reduced the open number of stomata (Figure 5B,G). Our results were nearly in line with Da costa et al. (2020), who reported that the higher concentration of CuO NPs reduced the size and number of stomata in plants [77]. The irregular and deformed stomata under CuO NPs stress were the consequence of ROS accumulation, which led to induced stomatal damage in rice seedlings (Figure 4A). The decreased stomatal damage in BR-treated seedlings with CuO NPs could be linked with a lower rate of ROS accumulation and oxidative damage in these seedlings. Plant stomatal openings depend on the concentration of 24-Epibrassinolide and the plant species under study. In tomato plants, the 24-Epibrassinolide (EBR) with a lower concentration induced stomatal opening; however, at higher levels, it mediated stomatal closure [91]. In contrast, ethylene (ACC) was found to actively participate in stomatal and guard cell damage under CuO NPs stress (Figure 5C,H), which further provides evidence that ethylene promotes ROS generation under CuO NPs and induces negative effects on guard cells and stomatal aperture. Interestingly, one of the studies showed that ethylene accelerated ROS production and damaged the stomatal aperture by regulating AtrbohF [92]. Our findings were also supported by Ali et al. (2017), who reported that ethylene took part in more ROS generation, leading to the destruction of guard cells and stomatal aperture in Arabidopsis thaliana leaves under BPA stress [49]. Furthermore, BRs showed an antagonistic response with ethylene (ACC) and mitigated the toxic effects of ACC on stomatal damage in rice seedlings (Figure 5D,I). These results indicated that BRs might inhibit ethylene biosynthesis and reduce the ethylene-induced toxic effects on the stomatal aperture (Figure 5E,J). Previously, one study reported that the inhibitory effect of BRs on ethylene biosynthesis led to mitigating the toxic effects of salinity in lettuce [54].
To overcome extra ROS production, plants develop an antioxidant enzyme system to protect cellular damage against stressful conditions [65]. The impact of NPs on the antioxidant defense system of plants mainly depends on the exposure time, NP concentrations, and the genotype of the plant species [93]. It has been reported that catalase (CAT) and ascorbate (APX) play an important role in the scavenging of ROS production [94]. CuO NPs significantly reduced the antioxidant enzyme activities in rice seedlings. Previously, different reports illustrated that NPs reduced the activities of enzymes in rice seedlings [86,95]. However, these enzyme activities were reduced in ACC-treated seedlings (Figure 8A–D). Previous studies also reported the involvement of ethylene in the reduction in the antioxidant enzyme system in Arabidopsis and rice under BPA and ZnO NPs stress, respectively [49,86]. Plants under CuO NPs toxicity in the presence of exogenous BRs exhibited more antioxidant enzyme activities as compared to the CuO NPs-treated seedlings alone (Figure 8A–D). The mechanism by which exogenous BRs restrict the level of ROS and MDA induced under CuO NPs might be due to the activation of the antioxidant defense system via BRs under stressful conditions [96]. These findings also align with the previous research indicating that BRs augment antioxidant responses in pepper plants exposed to Cr stress [97]. BRs showed an antagonistic response with ethylene under CuO NPs stress; they enhanced the activity of antioxidant enzymes in both roots and shoots (Figure 8A–D). This indicates that BRs may mitigate ethylene-induced CuO NPs oxidative stress in rice seedlings by improving the antioxidant defense system, possibly via controlling ethylene-induced negative effects on the antioxidant defense system. Likewise, in previous studies, it has been reported that BRs act antagonistically with ethylene-reduced ethylene biosynthesis and mitigate ethylene-induced negative effects on ascorbic acid (AA) (which improves the antioxidant defense system) in tomato plants [55]. These results demonstrate that BRs antagonistically interact with ethylene, which is also consistent with the negative interaction of ethylene–brassinosteroids in growth responses [98].

5. Conclusions

The present study illustrated that CuO NPs induced toxicity in rice seedlings by reducing plant biomass and growth, damaging the cellular ultrastructure and stomatal aperture. CuO NPs also increased the ROS accumulation and reduced the antioxidant defense system. Notably, the exogenous ethylene biosynthesis precursor (ACC) further enhanced the CuO NPs-induced toxicity on rice seedlings, reduced the antioxidant defense system, and damaged the cellular ultrastructure and stomatal aperture. On the contrary, the exogenous application of BRs actively reduced the CuO NPs’ phytotoxic effects and restored guard cells, stomatal aperture, and antioxidant defense systems against CuO NPs stress. Interestingly, exogenous BRs and ethylene showed antagonistic crosstalk under CuO NPs stress via partially reducing the ethylene-induced CuO NPs toxicity in rice seedlings. These findings have crucial application value since they illustrate the positive potential of BRs in mitigating CuO NPs toxicity, show the antagonistic crosstalk of BRs with ethylene, and overcome the ethylene-induced CuO NPs toxicity. These studies uncover the interesting crosstalk of two main phytohormones under CuO NPs stress, providing basic knowledge to improve crop yield and productivity in CuO NPs-contaminated areas. Figure 9 shows the schematic representation of how exogenous ethylene biosynthesis precursor (ACC) accelerates ROS accumulation and induces ultrastructural damages. This study provides valuable insights for agricultural scientists to optimize phytohormones (ethylene and BRs) pathways to mitigate CuO NP toxicity in crops. However, further investigation, including the effects of BRs on ethylene biosynthesis under CuO NPs stress through advanced molecular, genetic, and pharmacological approaches, may provide the complete crosstalk of BRs with ethylene under CuO NP stress in different crops.

Author Contributions

W.A.: Investigation, Data curation, Methodology, Writing—original draft. A.S.: Investigation, Methodology, Review and editing. A.R.K.: Investigation, Methodology, Review and editing. I.A.: Investigation. Y.G.: Funding acquisition, Supervision, Visualization, Conceptualization, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

We thank Zhenyu Qi from the Agricultural Experiment Station, Zhejiang University for their technical assistance during the experiment.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Characterization of CuO NPs. (A) Scanning electron microscopic (SEM) images of CuO NPs (B) and (C) EDX spectra. (D–G) Elemental mapping analysis of CuO NPs.
Figure 1. Characterization of CuO NPs. (A) Scanning electron microscopic (SEM) images of CuO NPs (B) and (C) EDX spectra. (D–G) Elemental mapping analysis of CuO NPs.
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Figure 2. Toxic effect of CuO NPs on rice growth. (A) Phenotypic changes in rice seedlings, (B) fresh weight per plant, (C) shoot length, (D) root length of rice seedlings exposed to CuO NPs (0 and 450 mg/L), ACC-CuO NPs, and BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
Figure 2. Toxic effect of CuO NPs on rice growth. (A) Phenotypic changes in rice seedlings, (B) fresh weight per plant, (C) shoot length, (D) root length of rice seedlings exposed to CuO NPs (0 and 450 mg/L), ACC-CuO NPs, and BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
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Figure 3. Effect of CuO NPs on chlorophyll content in rice seedlings. (A) Chlorophyll a and (B) chlorophyll b under different treatments of CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], and BR-ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
Figure 3. Effect of CuO NPs on chlorophyll content in rice seedlings. (A) Chlorophyll a and (B) chlorophyll b under different treatments of CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], and BR-ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
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Figure 4. Effect of CuO NPs on ROS accumulation and lipid peroxidation of rice. (A) NBT and DAB staining. (B,C) Quantification of NBT and DAB staining intensity estimated using ImageJ software (1.54p). (D) MDA content in shoots. (E) MDA content in roots under different treatments of CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
Figure 4. Effect of CuO NPs on ROS accumulation and lipid peroxidation of rice. (A) NBT and DAB staining. (B,C) Quantification of NBT and DAB staining intensity estimated using ImageJ software (1.54p). (D) MDA content in shoots. (E) MDA content in roots under different treatments of CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
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Figure 5. Scanning electron microscopy of leaf mesophyll cells of rice seedlings subjected to different treatments. CK (A,F), CuO NPs (450 mg/L) (B,G), ACC-CuO NPs (C,H), BR-CuO NPs (D,I), and BR-ACC-CuO NPs (E,J) [0.01 µM, 20 µM, and 450 mg/L]. Arrows indicate the stomata located on the leaf mesophyll cells of rice seedlings.
Figure 5. Scanning electron microscopy of leaf mesophyll cells of rice seedlings subjected to different treatments. CK (A,F), CuO NPs (450 mg/L) (B,G), ACC-CuO NPs (C,H), BR-CuO NPs (D,I), and BR-ACC-CuO NPs (E,J) [0.01 µM, 20 µM, and 450 mg/L]. Arrows indicate the stomata located on the leaf mesophyll cells of rice seedlings.
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Figure 6. Effect of BRs, ethylene, and CuO NPs on the ultrastructure of rice seedlings. (A) CK, (B) 450 mg/L, (C) ACC-CuO NPs, (D) BR-CuO NPs, (E) BR-ACC-CuO NPs. CW (cell wall), CM (cell membrane), Thy (thylakoids), M (mitochondria), NM (nuclear membrane), N (nucleus), NUE (nucleoli), PG (plastoglobuli), SG (starch grains).
Figure 6. Effect of BRs, ethylene, and CuO NPs on the ultrastructure of rice seedlings. (A) CK, (B) 450 mg/L, (C) ACC-CuO NPs, (D) BR-CuO NPs, (E) BR-ACC-CuO NPs. CW (cell wall), CM (cell membrane), Thy (thylakoids), M (mitochondria), NM (nuclear membrane), N (nucleus), NUE (nucleoli), PG (plastoglobuli), SG (starch grains).
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Figure 7. Scanning electron microscopy of rice root surface. Effects of different treatments on the root surface of rice seedlings. (A) CK, (B) CuO NPs 450 mg/L, (C) ACC-CuO NPs, (D) BR-CuO NPs, (E) BR-ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L].
Figure 7. Scanning electron microscopy of rice root surface. Effects of different treatments on the root surface of rice seedlings. (A) CK, (B) CuO NPs 450 mg/L, (C) ACC-CuO NPs, (D) BR-CuO NPs, (E) BR-ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L].
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Figure 8. Effect of CuO NPs on antioxidant enzyme activities of rice seedlings. (A) CAT activity in shoots; (B) CAT activity in roots; (C) APX activity in shoots; (D) APX activity in roots exposed to CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
Figure 8. Effect of CuO NPs on antioxidant enzyme activities of rice seedlings. (A) CAT activity in shoots; (B) CAT activity in roots; (C) APX activity in shoots; (D) APX activity in roots exposed to CuO NPs (0 and 450 mg/L), ACC-CuO NPs, BR-CuO NPs [0.01 µM and 450 mg/L], BRs, and ACC-CuO NPs [0.01 µM, 20 µM, and 450 mg/L]. In the figures, the error bars show ±SE and different letters indicate the significant difference at the probability (p < 0.05 by applying one-way ANOVA with the LSD test).
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Figure 9. Schematic representation of how exogenous ethylene biosynthesis precursor (ACC) accelerates ROS accumulation and induces ultrastructural damages. In contrast, brassinosteroids (BRs) inhibit CuO NP-induced toxicity. Interestingly, when BRs are applied in combination with ACC + CuO NPs, they suppress the ethylene-induced CuO NP toxicity on rice ultrastructural, stomatal, and root texture damage in rice seedlings.
Figure 9. Schematic representation of how exogenous ethylene biosynthesis precursor (ACC) accelerates ROS accumulation and induces ultrastructural damages. In contrast, brassinosteroids (BRs) inhibit CuO NP-induced toxicity. Interestingly, when BRs are applied in combination with ACC + CuO NPs, they suppress the ethylene-induced CuO NP toxicity on rice ultrastructural, stomatal, and root texture damage in rice seedlings.
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Azhar, W.; Salam, A.; Khan, A.R.; Ahmad, I.; Gan, Y. Brassinosteroids Alleviate Ethylene-Induced Copper Oxide Nanoparticle Toxicity and Ultrastructural and Stomatal Damage in Rice Seedlings. Agriculture 2025, 15, 907. https://doi.org/10.3390/agriculture15080907

AMA Style

Azhar W, Salam A, Khan AR, Ahmad I, Gan Y. Brassinosteroids Alleviate Ethylene-Induced Copper Oxide Nanoparticle Toxicity and Ultrastructural and Stomatal Damage in Rice Seedlings. Agriculture. 2025; 15(8):907. https://doi.org/10.3390/agriculture15080907

Chicago/Turabian Style

Azhar, Wardah, Abdul Salam, Ali Raza Khan, Irshan Ahmad, and Yinbo Gan. 2025. "Brassinosteroids Alleviate Ethylene-Induced Copper Oxide Nanoparticle Toxicity and Ultrastructural and Stomatal Damage in Rice Seedlings" Agriculture 15, no. 8: 907. https://doi.org/10.3390/agriculture15080907

APA Style

Azhar, W., Salam, A., Khan, A. R., Ahmad, I., & Gan, Y. (2025). Brassinosteroids Alleviate Ethylene-Induced Copper Oxide Nanoparticle Toxicity and Ultrastructural and Stomatal Damage in Rice Seedlings. Agriculture, 15(8), 907. https://doi.org/10.3390/agriculture15080907

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