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MAP  >  Fruit Research
2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Plant materials, growth conditions, and stress treatment
Vector construction
Chlorophyll and anthocyanin content determination
Physiological analysis
RNA isolation and qRT-PCR analysis
Results
Screening and characterization of lncRNAs
Generation of lncRNA overexpression lines
Effect of lncRNA overexpression on heat tolerance in Arabidopsis
Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis
Effect of lncRNA overexpression on drought tolerance in Arabidopsis
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
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References
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ARTICLE   Open Access    

Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit

  • 1.

    Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

  • 2.

    College of Horticulture, South China Agricultural University, Guangzhou 510642, China

  • 3.

    Bamboo Research Institute, Nanjing Forestry University, Nanjing 210037, China

  • 4.

    Department of Agriculture, University of Agriculture, Faisalabad 38000, Pakistan

  • 5.

    New Uzbekistan University, Mustaqillik Ave. 54, Tashkent 100007, Uzbekistan

  • 6.

    Anhui Dabieshan Academy of Traditional Chinese Medicine, Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resources, Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine, College of Biological and Pharmaceutical Engineering, West Anhui University, Lu'an 237012, China

  • 7.

    Biology Department, College of Science, King Khalid University, Abha 61413, Saudi Arabia

  • 8.

    Botany Section, School of Sciences, Maulana Azad National Urdu University, Hyderabad 500032, India

  • 9.

    School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

  • 10.

    Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW 2751, Australia

  • # Authors contributed equally: Iftikhar Hussain Shah, Irfan Ali Sabir

More Information
  • Received: 08 November 2023
    Revised: 01 March 2024
    Accepted: 12 March 2024
    Published online: 03 June 2024
    Fruit Research  4,  Article number: e022 (2024)  |  Cite this article
  • Nanotechnology has emerged as a prominent field in recent times. The fabrication of biocompatible materials has taken on highlighted significance owing to their requisite application in diverse sectors including medicine, water treatment and purification, health, and other related fields. There has been a lot of research done recently on the green synthesis of various nanoparticles (NPs). Copper a high-performance metal used in agriculture to combat pathogenic attacks, has received less attention. The current work demonstrates the successful preparation of green synthesized copper oxide nanoparticles (CuO.NPs) from Mangifera indica (M. indica) leaf extract. The spectral and morphological characterization biosynthesized were observed using, FTIR, XRD, and TEM analysis. The FTIR analysis revealed the functional groups present in plant extracts. XRD was carried out to demonstrate the crystalline nature and size of nanoparticles using the Scherrer formula. UV was performed to observe the optical properties of NPs. Further, Transmission electron spectroscopy (TEM) was carried out to confirm the physical shape of CuO.NPs with 50 nm. The M. indica mediated NPs were evaluated against gram-negative and positive bacteria Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) at different concentrations. The in vivo fungicidal activity was performed against Rhizophus oryzae (R. oryzae) on Diospyros kaki (persimmon fruit). The detach fruit method was applied to evaluate the potential of NPs. Higher exposure of 100 µg·mL−1 CuO.NPs showed mycelia inhibition followed by 30, 60, and control treatments. Furthermore, Green CuO.NPs showed prominent antioxidant activities as compared to plant sources. The findings obtained suggest that the green-formulated CuO.NPs could be further investigated for the treatment of many phytopathogenic diseases.

  • Introduction

    Plants are continuously subjected to unpredictable environmental conditions and encounter a multitude of stressors throughout their growth and development, posing a significant challenge to global crop production and food security[1]. Heat and drought are undoubtedly the two most important stresses that have a huge impact on crops. Both elicit a wide array of biochemical, molecular, and physiological alterations and responses, impacting diverse cellular processes and ultimately influencing crop yield and quality[2].

    A primary physiological consequence of both stresses is the diminished photosynthetic capacity, partially resulting from the degradation of chlorophyll due to leaf senescence under stress conditions. Chlorophyll accumulation was diminished in numerous plants subjected to drought or heat stress conditions[3,4]. Various environmental stresses prompt excessive generation of reactive oxygen species (ROS), initiating oxidative damage that compromises lipids, and proteins, and poses a serious threat to cellular functions[2]. To mitigate oxidative stress and minimize damage, plants have developed various protective mechanisms to neutralize ROS. Several antioxidant enzymes, such as SOD, POD, and CAT, are integral to cellular antioxidative defense mechanisms. Additionally, antioxidants such as anthocyanins and proline serve as crucial ROS scavengers[5,6]. The elevation in temperature typically induces the transient synthesis of heat shock proteins (Hsps), which function as molecular chaperones in protecting proteins from denaturation and aggregation, with their activity primarily regulated at the transcriptional level by heat shock factors (Hsfs)[7]. The significance of Hsps and Hsfs in all organisms, including plants, has been assessed in various stress conditions that could disrupt cellular homeostasis and result in protein dysfunction[7]. Drought stress can also trigger the transcription of a suite of marker genes, including RD29A, RD29B, NCED3, AREB1, Rab18, etc., which assist plants in mitigating cellular damage during dehydration and bolstering their resilience to stress[8−10].

    Previous research efforts focusing on the regulatory control of stress-related genes have largely centered around protein-coding genes. In recent years, non-protein-coding transcripts have emerged as important regulatory factors in gene expression. Among them, long non-coding RNAs (lncRNAs) lncRNAs have been identified as implicated in various abiotic stresses[11,12]. LncRNAs are a class of non-coding RNAs (ncRNAs) exceeding 200 nucleotides in length. They possess minimal or no protein-coding potential[13]. In plants, lncRNAs are specifically transcribed by RNA polymerases Pol IV, Pol V, Pol II, and Pol III[14,15]. LncRNAs exhibit low abundance and display strong tissue and cellular expression specificity relative to mRNAs. Moreover, sequence conservation of lncRNAs is was very poor across different plant species[13,16,17]. The widespread adoption of high-throughput RNA sequencing technology has revealed lncRNAs as potential regulators of plant development and environmental responses. In cucumber, RNA-seq analysis has predicted 2,085 lncRNAs to be heat-responsive, with some potentially acting as competitive endogenous RNAs (ceRNAs) to execute their functions[18]. In radish, a strand-specific RNA-seq (ssRNA-seq) technique identified 169 lncRNAs that were differentially expressed following heat treatment[19]. In Arabidopsis, asHSFB2a, the natural antisense transcript of HSFB2a was massively induced upon heat stress and exhibited a counteracted expression trend relative to HSFB2a. Overexpression of asHSFB2a entirely suppressed the expression of HSFB2a and impacted the plant's response to heat stress[20]. For drought stress resistance, 244 lncRNAs were predicted in tomatoes to be drought responsive probably by interacting with miRNAs and mRNAs[21]. Under drought stress and rehydration, 477 and 706 lncRNAs were differentially expressed in drought-tolerant Brassica napus Q2 compared to drought-sensitive B. napus, respectively[22]. In foxtail millet and maize, 19 and 644 lncRNAs, respectively, were identified as drought-responsive[23,24]. Despite the identification of numerous lncRNAs by high-throughput sequencing, which suggests their potential involvement in various abiotic stress processes, only a minority have been experimentally validated for function.

    In our previous study, we characterized 1,229 differentially expressed (DE) lncRNAs in Chinese cabbage as heat-responsive, and subsequent bioinformatics analysis reduced this number to 81, which are more likely associated with heat resistance[25]. lnc000283 and lnc012465 were selected from among them for further functional investigation. The findings indicated that both lnc000283 and lnc012465 could be promptly induced by heat shock (HS). Overexpression of either lnc000283 or lnc012465 in Arabidopsis plants enhanced their capacity to tolerate heat stress. Additionally, both lnc000283 and lnc012465 conferred drought tolerance to transgenic Arabidopsis.

    Materials and methods

    Plant materials, growth conditions, and stress treatment

    The lncRNA sequences examined in this study were from Chiifu-401-42 Chinese cabbage and all Arabidopsis plants were of the Col-0 background. Transgenic plants expressing lnc000283 and lnc012465 were generated using the Agrobacterium tumefaciens-mediated floral dip method[26]. Single-copy and homozygous T3 plants were identified through genetic segregation on an agar medium supplemented with kanamycin. The T3 generation plants, or their homozygous progeny, were utilized in the experiments.

    For phenotypic assessment, Arabidopsis seeds were initially sown on filter paper moistened with ddH2O and placed in a 4 °C freezer for 2 d. Subsequently, they were evenly planted in nutrient-rich soil and transferred to a growth chamber operating a 16-h day/8-h night cycle, with day/night temperatures of 22 °C/18 °C and a light intensity of 250 μmol·m−2·s−1. After 10 d of growth, Arabidopsis plants with uniform growth were transferred to 50-hole plates. Arabidopsis plants grown in Petri dishes were firstly seed-sterilized and then sown on 1/2 MS medium supplemented with 10 g·L−1 sucrose. The seeds were then placed in a 4 °C refrigerator for 2 d in the dark before transferring them to a light incubator. The day/night duration was set to 16 h/8 h, the day/night temperature to 21 °C/18 °C, and the light intensity to 100 μmol·m−2·s−1.

    For heat treatment, 3-week-old seedlings were subjected to 38 °C for 4 d within a light incubator, subsequently transferred to their original growth conditions under the same light/dark cycles. For drought treatment, 3-week-old Arabidopsis seedlings were deprived of water for 10 d, followed by rehydration to facilitate a 2-d recovery period. Plants were photographed and surveyed both before and after treatment.

    Vector construction

    The lncRNA sequences (lnc000283 and lnc012465) were chemically synthesized based on RNA-seq data, with restriction sites for BamH1 and Kpn1 engineered upstream and downstream. The resultant lncRNA constructs were subcloned into the pCambia2301 binary vector, incorporating a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vectors were transformed into Escherichia coli TOP10 competent cells (Clontech), incubated at 37 °C overnight, after which single clones were selected for PCR verification, and the confirmed positive colonies were submitted for sequencing. Following verification, the correct plasmids were introduced into A. tumefaciens strain GV3101 using the freeze-thaw method and subsequently transformed into Arabidopsis wild-type (Col) plants.

    Chlorophyll and anthocyanin content determination

    To quantify the chlorophyll content, the aerial portions of wild-type and transgenic Arabidopsis plants, grown in Petri dishes were weighed, minced, and then subjected to boiling in 95% ethanol until fully decolorized. Aliquots of 200 μL from the extract were transferred to a 96-well plate and the absorbance at 663 nm and 645 nm was measured via spectrophotometry by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for WT and each transgenic line. Chlorophyll content was determined according to the formula of the Arnon method[27]: Chlorophyll a = (12.72A663 − 2.59A645) v/w, Chlorophyll b = (22.88A645 − 4.67A663) v/w, Total chlorophyll = (20.29A645 + 8.05A663) v/w.

    The quantification of anthocyanin was performed as follows: aerial parts of wild-type and transgenic Arabidopsis plants, cultivated in Petri dishes, were weighed and ground to powder in liquid nitrogen. Subsequently, the samples were incubated in 600 μL of acidified methanol (containing 1% HCl) at 70 °C for 1 h. Following this, 1 mL of chloroform was added, and the mixture was vigorously shaken to remove chlorophyll. The mixture was then centrifuged at 12,000 rpm for 5 min, after which the absorbance of the aqueous phase was determined at 535 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). Three biological replicates were analyzed for WT and each transgenic line. The relative anthocyanin content was calculated according to anthocyanin concentration and extraction solution volume. One anthocyanin unit is defined as an absorption unit at a wavelength of 535 nm in 1 mL of extract solution. In the end, the quantity was normalized to the fresh weight of each sample.

    Physiological analysis

    Three-week-old transgenic and WT A. thaliana plants, subjected to normal conditions or varying durations of heat or drought stress, were utilized for subsequent physiological assessments. All assays were performed in accordance with the method described by Chen & Zhang[28]. In brief, 0.1 g of fresh leaf tissue was homogenized in 500 μL of 100 mM PBS (pH 7.8) while chilled on ice. The homogenate was then centrifuged at 4 °C, and the resultant supernatant was employed for further analysis. For the determination of MDA content, 100 μL of the supernatant was combined with 500 μL of a 0.25% thiobarbituric acid (TBA) solution (which was prepared by dissolving 0.125 g of TBA in 5 mL of 1 mol·L−1 NaOH before being added to 45 mL of 10% TCA) and boiled for 15 min. Following a 5 min cooling period on ice, the absorbance was measured at 532 nm and 600 nm. The activity of POD was determined as follows: initially, 28 μL of 0.2% guaiacol and 19 μL of 30% H2O2 were sequentially added to 50 mL of 10mM PBS (pH 7.0), after thorough heating and mixing, 1 mL was transferred into a cuvette, then 50 μL of the supernatant was added to the cuvette and the absorbance at 470 nm was monitored every 15 s for 1 min. To determine the proline content, a reaction solution was prepared by mixing 3% sulfosalicylic acid, acetic acid, and 2.5% acidic ninhydrin in a ratio of 1:1:2, then 50 μL of the supernatant was added to 1 mL of the reaction solution, which was then subjected to a boiling water bath for 15 min (the solution turned red after the boiling water bath). Following cooling on ice, the absorbance at 520 nm was recorded. For the quantification of proline, an L-proline standard curve was prepared by dissolving 0, 5, 10, 15, 20, 25, and 30 μg of L-proline in 0.5 mL of ddH2O, followed by the addition of 1 mL of the reaction solution and measuring the absorbance at 520 nm. The proline content in the samples was then determined based on the L-proline standard curve.

    RNA isolation and qRT-PCR analysis

    Total RNA was isolated from the aerial parts of Arabidopsis using the TaKaRa MiniBEST Plant RNA Extraction Kit, followed by purification and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). The cDNA product was diluted 10 times and real-time PCR was conducted in triplicate for each biological replicate using SYBR PCR Master Mix (Applied Biosystems) on the ABI 7500 system under the following conditions: 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s and 60 °C for 30 s. The relative expression levels of each gene were normalized against the transcript abundance of the endogenous control UBC30 (At5g56150) and calculated using the 2−ΔCᴛ method. The specific primers employed for qRT-PCR are detailed in Supplemental Table S1.

    Results

    Screening and characterization of lncRNAs

    In our prior investigation, dozens of lncRNAs associated with the heat stress response in Chinese cabbage were identified through informatics analysis. Two lncRNAs (lnc000283 and lnc012465) were chosen for genetic transformation in Arabidopsis to elucidate their functions comprehensively. Transcriptome data analysis indicated that the expression of lnc000283 and lnc012465 in Chinese cabbage were both induced by HS. To verify the accuracy, the expression patterns of lnc000283 and lnc012465 were confirmed through quantitative real-time PCR (qRT-PCR), and the results from qRT-PCR were consistent with those obtained from RNA-seq (Fig. 1a). The corresponding homologous genes in Arabidopsis were identified as CNT2088434 and CNT2088742, exhibiting sequence similarities of 88% and 87%, respectively (Supplemental Fig. S1). Subcellular localization predictions using the lnclocator database (www.csbio.sjtu.edu.cn/bioinf/lncLocator) suggested that both lncRNAs are localized within the nucleus (Supplemental Table S2). Bioinformatics analysis was conducted using the CPC tool (http://cpc.cbi.pku.edu.cn/) indicated that lnc000283 and lnc012465 are noncoding sequences, with coding probabilities of 0.0466805 and 0.0432148, respectively comparable to the well-characterized lncRNAs COLDAIR and Xist, but significantly lower than those of the protein-coding genes UBC10 and ACT2 (Fig. 1b).

    Figure 1.  Characteristics of lnc000283 and lnc012465. (a) Expression level of lnc000283 and lnc012465 in Chinese cabbage leaves treated at 38 °C at different time points, as determined by qRT-PCR and RNA-seq. CK is a representative plant before heating, and T1, T4, T8, and T12 denote plants that were subjected to 38 °C for 1, 4, 8, and 12 h, respectively. The expression levels were normalized to the expression level of Actin. (b) Analysis of coding potential for lnc000283 and lnc012465. The coding potential scores were calculated using the CPC program. UBC10 (At5g53300) and ACT2 (At3g18780) are positive controls that encode proteins. COLDAIR (HG975388) and Xist (L04961) serve as negative controls, exhibiting minimal protein-coding potential.
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    Generation of lncRNA overexpression lines

    To elucidate the role of lnc000283 and lnc012465 in response to abiotic stress, overexpression vectors were constructed for these lncRNAs, driven by the CaMV 35S promoter, and they were introduced into Arabidopsis thaliana (Col-0 ecotype). Through PCR identification and generational antibiotic screening, two homozygous positive lines for lnc012465 and lnc000283 were obtained. The relative expression levels of these lncRNAs were assessed using qRT-PCR (Fig. 2a). When plants were grown in 1/2 MS medium, with the consumption of nutrients, and reduction of water, the leaves of WT began to turn yellow, but the lnc000283 and lnc012465 overexpression lines developed a deep purple color of leaf veins (Fig. 2b). Examination of chlorophyll and anthocyanin contents in the plants revealed that both overexpression lines had higher levels of chlorophyll and anthocyanin compared to the WT, suggesting that the transgenic plants might possess enhanced resistance to nutritional or water stress (Fig. 2c, d).

    Figure 2.  Arabidopsis plants overexpressing lnc000283 and lnc012465 had higher anthocyanins and chlorophyll content. (a) The relative expression level of lnc000283 and lnc012465 in WT and different transgenic lines. UBC10 (At5g53300) was used as an internal control. Each value is mean ± sd (n = 3). (b) The phenotype of WT and Arabidopsis overexpressing lnc000283 or lnc012465 grown on 1/2 MS medium 50 d after sowing. The (c) anthocyanin and (d) chlorophyll content of WT and transgenic Arabidopsis overexpressing lnc000283 or lnc012465. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01).
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    Effect of lncRNA overexpression on heat tolerance in Arabidopsis

    Given that lnc000283 and lnc012465 were highly induced by heat, the thermotolerance of the overexpressing (OE) plants were compared to that of the wild type. Arabidopsis plants were initially exposed to a an HS treatment at 38 °C for 4 d, followed by recovery at room temperature. The death caused by HS was processive. Post-severe HS challenge for 4 d, OE plants initially appeared similar to WT, but upon recovery, their leaves started to fold or curl, followed by a transition to yellow, white, and eventually drying out (Fig. 3a). OE lnc000283 and OE lnc012465 plants exhibited enhanced thermotolerance compared to WT, with lnc012465 showing particularly strong tolerance (Fig. 3a; Supplemental Fig. S2a). After 5 d of recovery, leaf coloration indicated that transgenic plants maintained a significantly higher percentage of green leaves and a lower percentage of bleached leaves compared to WT (Fig. 3b; Supplemental Fig. S2b). Under non-heat-stress conditions, WT and OE plants possessed comparable water content. However, following heat stress, the fresh-to-dry weight ratio of OE lnc000283 and lnc012465 lines was significantly greater than that of WT (Fig. 3c; Supplemental Fig. S2c). Abiotic stresses frequently trigger the production of excessive reactive oxygen species (ROS), which are believed to cause lipid peroxidation of membrane lipids, leading to damage to macromolecules. Leaf MDA content is commonly used as an indicator of lipid peroxidation under stress conditions; therefore, the MDA content in both transgenic and WT plants was assessed. Figure 3d shows that the MDA content in WT plants progressively increased after heat treatment, whereas in the two lines overexpressing lnc012465, the MDA content increased only slightly and remained significantly lower than that in WT at all time points. In plants overexpressing lnc000283, the MDA content did not significantly differ from that of WT before heat stress. However, after 4 d of heat treatment, the MDA content was significantly lower compared to WT (Supplemental Fig. S2d). The results suggested that the expression of both lnc012465 and lnc000283 can mitigate injury caused by membrane lipid peroxidation under heat-stress conditions. Peroxidase (POD) is a crucial antioxidant enzyme involved in ROS scavenging. Figure 3e and Supplemental Fig. S2e demonstrate that POD activity increased in both transgenic and WT plants after heat treatment. However, the increase in WT plants was modest, whereas OE lnc000283 and OE lnc012465 plants exhibited consistently higher POD activity. As anticipated, proline levels were induced in response to stress in all studied plants (Fig. 3f; Supplemental Fig. S2f). However, under normal conditions and 2 d post-heat stress treatment, the proline content in OE lnc000283 and OE lnc012465 plants did not exhibit significant changes compared to WT (Fig. 3f; Supplemental Fig. S2f). Moreover, after 4 d of heat stress, the proline content in OE lnc012465 lines was significantly lower than in WT, and the OE lnc000283 transgenic line 12-6 also showed a marked decrease in proline content compared to WT (Fig. 3f; Supplemental Fig. S2f). The results indicated that the thermotolerance of plants overexpressing either lnc000283 or lnc012465 was independent of proline accumulation.

    Figure 3.  Overexpressing lnc012465 lines are more tolerant to heat stress. (a) Phenotypes of WT and OE lnc012465 plants were assessed before and after exposure to heat stress. The heat treatment was applied to 25-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh-to-dry weight ratio of Arabidopsis leaves was measured before and after 38 °C heat treatment. (d)−(f) depict the MDA content, POD activity, and proline content in Arabidopsis leaves at varying durations of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis

    To elucidate the molecular mechanisms by which lncRNAs enhance thermotolerance in Arabidopsis, the expression of the Hsf gene HsfA7a and three Hsps (Hsp25.3, Hsa32, and Hsp18.1-CI) in OE lnc000283, OE lnc012465, and WT Arabidopsis plants were investigated at various time points following heat treatment. As shown in Fig. 4 and Supplemental Fig. S3, both Hsf and Hsps exhibited a rapid response to heat stress with strong induction. Notably, the transcripts of HsfA7a and Hsp25.3 were significantly upregulated at 1 h after heat exposure, then experienced a sharp decrease. Hsa32 and Hsp18.1-CI were highly induced at 1 h and, unlike the other proteins, sustained high expression levels at 3 h (Fig. 4; Supplemental Fig. S3). At 1 h post-heat treatment, the transcript levels of Hsa32 and HsfA7a in OE lnc000283 did not significantly differ from those in WT. However, by 3 h, Hsa32 expression was roughly 50% of the WT level, while HsfA7a expression was approximately double that of WT (Supplemental Fig. S3). The overexpression of lnc000283 did not significantly affect the transcript level of Hsp25.3 at any of the tested time points. Notably, Hsp18.1-CI expression in both lines overexpressing lnc000283 was significantly induced at all three detection points post-heat treatment, reaching approximately 4-9-fold higher levels than in the WT (Supplemental Fig. S3). In Arabidopsis plants with elevated expression of lnc012465, the expression patterns of all Hsp and Hsf genes were similar to those in plants overexpressing lnc000283, with the notable exception of Hsa32. Unlike the WT, Hsa32 did not show a trend of down-regulation at 3 h post-heat treatment (Fig. 4). The findings suggest that the substantial induction of Hsp18.1-CI may play a role in enhancing the thermotolerance of Arabidopsis plants overexpressing lnc000283 and lnc012465.

    Figure 4.  The expression of HSF and HSP genes in lnc012465 overexpressing lines before and after different heat treatment times. Gene expression levels were quantified using RT-qPCR and normalized to UBC10 (At5g53300). Each value represents the mean ± standard deviation (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on drought tolerance in Arabidopsis

    Prior research has implicated a significant proportion of genes in conferring resistance to various abiotic stresses. To elucidate the functions of lnc000283 and lnc012465 more thoroughly, WT and transgenic plants were subjected to drought stress by depriving them of water for 9 d. It was noted that the majority of leaves in WT plants withered and dried, whereas the OE lnc000283 and OE lnc012465 plants exhibited reduced withering, with only a minority displaying dryness (Fig. 5a; Supplemental Fig. S4a). Eight days post-rewatering, a negligible fraction of WT seedlings exhibited recovery, whereas the overwhelming majority of transgenic plants regained vigorous growth (Fig. 5a; Supplemental Fig. S4a). The transgenic plants demonstrated a significantly higher survival rate compared to the WT plants. Following 9 d of water deficit treatment, less than 40% of the WT plants survived, whereas the OE 012465 lines 8-7 and 9-1 exhibited survival rates of 100% and 95%, respectively, and the OE 000283 lines 11-10 and 12-6 had survival rates of 87% each. (Fig. 5b; Supplemental Fig. S4b). Water loss serves as a critical metric for assessing plant drought tolerance, hence the fresh-to-dry weight ratio of excised leaves was assessed via desiccation analysis. Following 4 d of drought treatment, the fresh-to-dry weight ratio for WT plants was reduced to 43%, whereas for OE lnc000283 lines 11-10 and 12-6, it was reduced to 73% and 75%, respectively. For OE 012465 lines 8-7 and 9-1, the ratios were reduced to 67% and 62%, respectively (Fig. 5c; Supplemental Fig. S4c). The findings indicated that lnc000283 and lnc012465 endow the transgenic plants with drought tolerance.

    Figure 5.  Overexpressing lnc012465 lines are more tolerant to drought stress. (a) Phenotype of WT and OE lnc012465 plants before and after subjecting to drought stress. Drought treatment was carried out on 20-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh weight to dry weight ratio of Arabidopsis leaves before and after undergoing 38 °C heat treatment. (d)−(f) MDA content, POD activity, and proline content in Arabidopsis leaves under different times of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
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    MDA content in leaves is a standard biomarker for assessing the extent of drought stress-induced damage. Prior to drought stress exposure, MDA levels in WT and transgenic plants were comparable. However, following 7 and 9 d of water deficit, the MDA content in the transgenic plants was markedly reduced compared to the WT, suggesting a less severe degree of membrane lipid peroxidation in the transgenic plants (Fig. 5d; Supplemental Fig. S4d). Oxidative stress frequently coincides with drought stress, hence the activity of POD was assessed to evaluate the ROS scavenging ability. The findings indicated that as the duration of drought treatment increased, POD activity progressively rose. Before drought exposure, the POD activity in lines 11-10 and 12-6 of OE 000283 was 2.4-fold and 2.2-fold higher than that of the WT, respectively (refer to Supplemental Fig. S4e). Following drought treatment, the POD activity in the transgenic lines remained significantly elevated compared to the wild type, although the enhancement was less pronounced than before the treatment (Supplemental Fig. S4e). In the OE 012465 plants, the POD activity in lines 8-7 and 9-1 significantly surpassed that of the wild type, with the discrepancy being more pronounced during drought stress (Fig. 5e). The proline content in WT and OE 000283 plants exhibited no significant differences before and after 7 d of treatment. However, after 9 d of drought, the proline content in OE 000283 plants was significantly lower compared to that in the WT (Supplemental Fig. S4f). OE 000465 plants showed no significant difference from the wild type before and after drought treatment (Fig. 5f). The findings were consistent with those under heat stress, indicating that the enhanced stress resistance due to the overexpression of lnc000283 and lnc012465 in Arabidopsis is not reliant on proline accumulation.

    Following drought stress treatment, the expression levels of drought-related genes such as RD29A, RD29B, NCED3, AREB1, and Rab18 were significantly elevated in plants overexpressing lnc000283 and lnc012465 compared to WT plants. These findings suggest that lnc000283 and lnc012465 modulate Arabidopsis drought tolerance by regulating the expression of genes associated with the drought stress response (Fig. 6; Supplemental Fig. S5).

    Figure 6.  The expression of drought-responsive genes in lnc012465 overexpressing lines before and after different drought treatment time. Gene expression levels were determined by qRT-PCR normalized against UBC10 (At5g53300). Each value is mean ± sd (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Discussion

    The integrity of global food security is under threat due to the confluence of rapid population expansion and profound climatic shifts[29]. Amidst the shifting climatic landscape, heat and drought stress have emerged as primary limitations to crop yield and global food security. Understanding how plants detect stress cues and acclimate to challenging conditions is a pivotal biological inquiry. Moreover, enhancing plant resilience to stress is essential for maintaining agricultural productivity and fostering environmental sustainability[2]. Concurrently, the advancement of next-generation sequencing (NGS) technology has led to the identification of a substantial number of lncRNAs that participate in diverse stress responses, with functional analyses having been conducted on several of these molecules.[30] For instance, in the case of potatoes, the lncRNA StFLORE has been identified to modulate water loss through its interaction with the homologous gene StCDF1[31]. LncRNA TCONS_00021861 can activate the IAA biosynthetic pathway, thereby endowing rice with resistance to drought stress[32]. In wheat, the expression of TalnRNA27 and TalnRNA5 was upregulated in response to heat stress[33]. Our prior investigation identified a total of 81 lncRNAs in Chinese cabbage that engage in intricate interactions with their respective mRNA targets across various phases of heat treatment[25]. Two lncRNAs, lnc000283 and lnc012465, were chosen for subsequent functional analysis. Findings confirmed that these lncRNAs endow transgenic Arabidopsis plants with enhanced tolerance to both heat and drought, thereby offering novel resources for enhancing stress resistance through genetic engineering.

    Abiotic stresses frequently trigger the synthesis of anthocyanins, serving as natural antioxidants that mitigate oxidative damage by neutralizing surplus reactive oxygen species (ROS), thereby protecting plants from growth inhibition and cell death, allowing plants to adapt to abiotic stresses[34,35]. For instance, during chilling stress, the accumulation of anthocyanins within leaves can mitigate oxidative damage, thereby enhancing the photosynthetic rate[36]. Consequently, the level of abiotic stress tolerance can be inferred from the concentration of anthocyanins. The reduction of photosynthetic ability is one of the key physiological phenomena of stresses, which is partly due to the degradation of chlorophyll caused by leaf senescence during stress. The reduced accumulation of chlorophyll in the plants was seen in many plants when exposed to drought or heat stress conditions. The current investigation revealed that lncRNA-overexpressing plants cultivated in Petri dishes exhibited increased accumulation of both chlorophyll and anthocyanins in advanced growth phases, indicating that these transgenic plants, overexpressing lnc000283 and lnc012465, demonstrated enhanced stress tolerance and superior growth performance relative to WT (Fig. 2c, d).

    Upon exposure to heat stress, there is a marked induction of transcription for numerous genes that encode molecular chaperones in plants, with the vast majority of these genes contributing to the prevention of protein denaturation-related damage and the augmentation of thermotolerance[37−39]. The present investigation identified multiple heat-inducible genes in plants overexpressing lnc000283 and lnc012465, as well as in WT (Fig. 4; Supplemental Fig. S3). The findings indicated that of the four HSP or HSF genes examined, Hsp18.1-CI exhibited a significantly greater abundance in both OE lnc000283 and OE lnc012465 plants compared to the WT following heat treatment for several days. Hsp18.1-CI, formerly referred to as Hsp18.2 has been the subject of investigation since 1989.[40] Following the fusion of the 5' region of Hsp18.2 in frame with the uidA gene of Escherichia coli, the activity of GUS, serving as the driver gene was observed to increase upon exposure to HS[40]. The Arabidopsis hsfA2 mutant exhibited diminished thermotolerance after heat acclimation, with the transcript levels of Hsp18.1-CI being substantially reduced compared to those in wild-type plants following a 4-h recovery period[41]. The findings revealed that the upregulation of Hsp18.1-CI protein is a critical mechanism by which plants achieve enhanced protection against heat stress in adverse environmental conditions, thereby bolstering their thermotolerance.

    Plants cultivated in natural settings are often subjected to concurrent multiple abiotic stresses, which can exacerbate threats to their routine physiological functions, growth, and developmental processes[42,43]. Elucidating the molecular mechanisms underlying plant responses to abiotic stress is crucial for the development of new crop varieties with enhanced tolerance to multiple abiotic stresses. Previous research has indicated that the overexpression of certain protein-coding genes can endow plants with resistance to a variety of abiotic stresses. For instance, tomatoes with robust expression of ShCML44 demonstrated significantly enhanced tolerance to drought, cold, and salinity stresses[44]. Overexpression of PeCBF4a in poplar plants confers enhanced tolerance to a range of abiotic stresses[45]. With respect to lncRNAs, transgenic Arabidopsis plants that overexpress lncRNA-DRIR displayed marked increased tolerance to salt and drought stresses compared to the wild-type[46]. In the present study, both overexpression lines of lnc000283 and lnc012465 exhibited resistance to heat and drought stresses, thereby contributing to the enhancement of plant resilience against multiple stresses (Figs 3, 5; Supplemental Figs S2, S4).

    The number of genes implicated in plant drought resistance is regulated by both ABA-dependent and ABA-independent pathways[47,48]. It is well established that the expression of RD29A exhibits a high level of responsiveness to drought stress, operating through both ABA-dependent and ABA-independent mechanisms[49]. RD29B, AREB1, and RAB18 are governed by an ABA-dependent regulatory pathway[10,49,50]. NCED3 is involved in ABA biosynthesis[51]. In the present study, the transcript levels of RD29A, RD29B, NCED3, AREB1, and RAB18 were significantly elevated in OE lnc000283 and OE lnc012465 plants compared to those in the WT plants (Fig. 6; Supplemental Fig. S5). The findings indicated that the drought tolerance imparted by OE lnc000283 and OE lnc012465 plants is contingent upon an ABA-dependent mechanism.

    Prior research has indicated that certain long non-coding RNAs (lncRNAs) can assume analogous roles across diverse biological contexts. For example, the lncRNA bra-miR156HG has been shown to modulate leaf morphology and flowering time in both B. campestris and Arabidopsis[52]. Heterogeneous expression of MSL-lncRNAs in Arabidopsis has been associated with the promotion of maleness, and similarly, it is implicated in the sexual lability observed in female poplars[53]. In the present study, lnc000283 and lnc012465 were induced by heat in Chinese cabbage, and their heterologous expression was found to confer heat tolerance in Arabidopsis. Additionally, sequences homologous to lnc000283 and lnc012465 were identified in Arabidopsis (Supplemental Fig. S1). The data suggest that these sequences may share a comparable function to that of heat-inducible sequences, potentially accounting for the conservation of lnc000283 and lnc012465'os functionality across various species.

    Conclusions

    In conclusion, the functions of two heat-inducible lncRNAs, lnc000283 and lnc012465 have been elucidated. Transgenic Arabidopsis lines overexpressing these lncRNAs accumulated higher levels of anthocyanins and chlorophyll at a later stage of growth compared to the WT when grown on Petri dishes. Furthermore, under heat and drought stress conditions, these OE plants exhibited enhanced stress tolerance, with several genes related to the stress resistance pathway being significantly upregulated. Collectively, these findings offer novel insights for the development of new varieties with tolerance to multiple stresses.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and supervision: Li N, Song X; experiment performing: Wang Y, Sun S; manuscript preparation and revision: Wang Y, Feng X, Li N. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32172583), the Natural Science Foundation of Hebei (C2021209019), the Natural Science Foundation for Distinguished Young Scholars of Hebei (C2022209010), and the Basic Research Program of Tangshan (22130231H).

  • The authors declare that they have no conflict of interest.

  • [1]

    Ray DK, Mueller ND, West PC, Foley JA. 2013. Yield trends are insufficient to double global crop production by 2050. PLoS One 8:e66428

    doi: 10.1371/journal.pone.0066428

    CrossRef   Google Scholar

    [2]

    Gull A, Lone AA, Ul Islam Wani N. 2019. Biotic and abiotic stresses in plants. In Abiotic and Biotic Stress in Plants, ed. de Oliveira AB. IntechOpen. pp. 1−19. http://dx.doi.org/10.5772/intechopen.85832
    [3]

    Basit A, Shah ST, Ullah I, Ullah I, Mohamed HI. 2021. Microbial bioactive compounds produced by endophytes (bacteria and fungi) and their uses in plant health. In Plant Growth-Promoting Microbes for Sustainable Biotic and Abiotic Stress Management, eds Mohamed HI, El-Beltagi HEDS, Abd-Elsalam KA. Cham: Springer. pp. 285–318. https://doi.org/10.1007/978-3-030-66587-6_11
    [4]

    Tiwari RK, Lal MK, Naga KC, Kumar R, Chourasia KN, et al. 2020. Emerging roles of melatonin in mitigating abiotic and biotic stresses of horticultural crops. Scientia Horticulturae 272:109592

    doi: 10.1016/j.scienta.2020.109592

    CrossRef   Google Scholar

    [5]

    Jones RAC. 2021. Global plant virus disease pandemics and epidemics. Plants 10:233

    doi: 10.3390/plants10020233

    CrossRef   Google Scholar

    [6]

    Shuping DSS, Eloff JN. 2017. The use of plants to protect plants and food against fungal pathogens: a review. African Journal of Traditional, Complementary and Alternative Medicines 14:120−27

    doi: 10.21010/ajtcam.v14i4.14

    CrossRef   Google Scholar

    [7]

    Panda S, Zhou K. 2023. Engineering microbes to overproduce natural products as agrochemicals. Synthetic and Systems Biotechnology 8:79−85

    doi: 10.1016/j.synbio.2022.11.005

    CrossRef   Google Scholar

    [8]

    Bai Z, Schmidt-Traub G, Xu J, Liu L, Jin X, et al. 2020. A food system revolution for China in the post-pandemic world. Resources, Environment and Sustainability 2:100013

    doi: 10.1016/j.resenv.2020.100013

    CrossRef   Google Scholar

    [9]

    Cestonaro LV, Garcia SC, Nascimento S, Gauer B, Sauer E, et al. 2020. Biochemical, hematological and immunological parameters and relationship with occupational exposure to pesticides and metals. Environmental Science and Pollution Research 27:29291−302

    doi: 10.1007/s11356-020-09203-3

    CrossRef   Google Scholar

    [10]

    Carneiro HCS, Bastos RW, Ribeiro NQ, Gouveia-Eufrasio L, Costa MC, et al. 2020. Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii. Science of The Total Environment 740:140135

    doi: 10.1016/j.scitotenv.2020.140135

    CrossRef   Google Scholar

    [11]

    Das Kurmi B, Tekchandani P, Paliwal R, Rai Paliwal S. 2017. Transdermal drug delivery: opportunities and challenges for controlled delivery of therapeutic agents using nanocarriers. Current Drug Metabolism 18:481−95

    doi: 10.2174/1389200218666170222150555

    CrossRef   Google Scholar

    [12]

    Patel P, Shah J. 2017. Safety and toxicological considerations of nanomedicines: the future directions. Current Clinical Pharmacology 12:73−82

    doi: 10.2174/1574884712666170509161252

    CrossRef   Google Scholar

    [13]

    Amoabediny G, Haghiralsadat F, Naderinezhad S, Helder MN, Akhoundi Kharanaghi E, et al. 2018. Overview of preparation methods of polymeric and lipid-based (niosome, solid lipid, liposome) nanoparticles: a comprehensive review. International Journal of Polymeric Materials and Polymeric Biomaterials 67:383−400

    doi: 10.1080/00914037.2017.1332623

    CrossRef   Google Scholar

    [14]

    Wang J, Hu X, Xiang D. 2018. Nanoparticle drug delivery systems: an excellent carrier for tumor peptide vaccines. Drug Delivery 25:1319−27

    doi: 10.1080/10717544.2018.1477857

    CrossRef   Google Scholar

    [15]

    Yin IX, Zhang J, Zhao IS, Mei ML, Li Q, et al. 2020. The antibacterial mechanism of silver nanoparticles and its application in dentistry. International Journal of Nanomedicine 15:2555−62

    doi: 10.2147/IJN.S246764

    CrossRef   Google Scholar

    [16]

    Kuppusamy P, Yusoff MM, Maniam GP, Govindan N. 2016. Biosynthesis of metallic nanoparticles using plant derivatives and their new avenues in pharmacological applications–an updated report. Saudi Pharmaceutical Journal 24:473−84

    doi: 10.1016/j.jsps.2014.11.013

    CrossRef   Google Scholar

    [17]

    Liaquat F, Kim HS, Kim S, Alyan A, Shah IH, et al. 2023. Application of Trichoderma spp. combined with iron oxide nanoparticles improves the physiochemical response of Arabidopsis thaliana under drought. Journal of Plant Biology 66:395−405

    doi: 10.1007/s12374-023-09399-0

    CrossRef   Google Scholar

    [18]

    Stensberg MC, Wei Q, McLamore ES, Porterfield DM, Wei A, et al. 2011. Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine 6:879−98

    doi: 10.2217/nnm.11.78

    CrossRef   Google Scholar

    [19]

    Gharpure S, Akash A, Ankamwar B. 2020. A review on antimicrobial properties of metal nanoparticles. Journal of Nanoscience and Nanotechnology 20:3303−39

    doi: 10.1166/jnn.2020.17677

    CrossRef   Google Scholar

    [20]

    Bloom DE, Cadarette D. 2019. Infectious disease threats in the twenty-first century: strengthening the global response. Frontiers in Immunology 10:549

    doi: 10.3389/fimmu.2019.00549

    CrossRef   Google Scholar

    [21]

    Tan KB, Sun D, Huang J, Odoom-Wubah T, Li Q. 2021. State of arts on the bio-synthesis of noble metal nanoparticles and their biological application. Chinese Journal of Chemical Engineering 30:272−90

    doi: 10.1016/j.cjche.2020.11.010

    CrossRef   Google Scholar

    [22]

    Salem SS, Fouda A. 2021. Green synthesis of metallic nanoparticles and their prospective biotechnological applications: an overview. Biological Trace Element Research 199:344−70

    doi: 10.1007/s12011-020-02138-3

    CrossRef   Google Scholar

    [23]

    Hussain I, Singh NB, Singh A, Singh H, Singh SC. 2016. Green synthesis of nanoparticles and its potential application. Biotechnology Letters 38:545−60

    doi: 10.1007/s10529-015-2026-7

    CrossRef   Google Scholar

    [24]

    Asmathunisha N, Kathiresan K. 2013. A review on biosynthesis of nanoparticles by marine organisms. Colloids and Surfaces B: Biointerfaces 103:283−87

    doi: 10.1016/j.colsurfb.2012.10.030

    CrossRef   Google Scholar

    [25]

    Zulfiqar H, Zafar A, Rasheed MN, Ali Z, Mehmood K, et al. 2019. Synthesis of silver nanoparticles using Fagonia cretica and their antimicrobial activities. Nanoscale Advances 1:1707−13

    doi: 10.1039/C8NA00343B

    CrossRef   Google Scholar

    [26]

    Chandirika JU, Annadurai G. 2018. Biosynthesis and characterization of silver nanoparticles using leaf extract Abutilon indicum. Global Journal of Biotechnology & Biochemistry 13:7−11

    Google Scholar

    [27]

    Rajput VD, Minkina T, Suskova S, Mandzhieva S, Tsitsuashvili V, et al. 2018. Effects of copper nanoparticles (CuO NPs) on crop plants: a mini review. BioNanoScience 8:36−42

    doi: 10.1007/s12668-017-0466-3

    CrossRef   Google Scholar

    [28]

    Qasim Nasar M, Zohra T, Khalil AT, Saqib S, Ayaz M, et al. 2019. Seripheidium quettense mediated green synthesis of biogenic silver nanoparticles and their theranostic applications. Green Chemistry Letters and Reviews 12:310−22

    doi: 10.1080/17518253.2019.1643929

    CrossRef   Google Scholar

    [29]

    Parvez GM. 2016. Pharmacological activities of mango (Mangifera indica): a review. Journal of Pharmacognosy and Phytochemistry 5:1−7

    Google Scholar

    [30]

    Rojas R, Alvarez-Pérez OB, Contreras-Esquivel JC, Vicente A, Flores A, et al. 2020. Valorisation of mango peels: extraction of pectin and antioxidant and antifungal polyphenols. Waste and Biomass Valorization 11:89−98

    doi: 10.1007/s12649-018-0433-4

    CrossRef   Google Scholar

    [31]

    Sabir IA, Manzoor MA, Shah IH, Ahmad Z, Liu X, et al. 2024. Unveiling the effect of gibberellin-induced iron oxide nanoparticles on bud dormancy release in sweet cherry (Prunus avium L.). Plant Physiology and Biochemistry 206:108222

    doi: 10.1016/j.plaphy.2023.108222

    CrossRef   Google Scholar

    [32]

    Shah IH, Ashraf M, Sabir IA, Manzoor MA, Malik MS, et al. 2022. Green synthesis and Characterization of Copper oxide nanoparticles using Calotropis procera leaf extract and their different biological potentials. Journal of Molecular Structure 1259:132696

    doi: 10.1016/j.molstruc.2022.132696

    CrossRef   Google Scholar

    [33]

    Labulo AH, David OA, Terna AD. 2022. Green synthesis and characterization of silver nanoparticles using Morinda lucida leaf extract and evaluation of its antioxidant and antimicrobial activity. Chemical Papers 76:7313−25

    doi: 10.1007/s11696-022-02392-w

    CrossRef   Google Scholar

    [34]

    Wu S, Rajeshkumar S, Madasamy M, Mahendran V. 2020. Green synthesis of copper nanoparticles using Cissus vitiginea and its antioxidant and antibacterial activity against urinary tract infection pathogens. Artificial Cells, Nanomedicine, and Biotechnology 48:1153−58

    doi: 10.1080/21691401.2020.1817053

    CrossRef   Google Scholar

    [35]

    Xu L, Zhang L, Ren D, Peng Y, Liu Z, et al. 2022. Green synthesis of Cu/Fe3O4 nanoparticles using green tea extract: evaluation of its catalytic activity, antioxidant and anti-colon cancer effects. Inorganic Chemistry Communications 144:109927

    doi: 10.1016/j.inoche.2022.109927

    CrossRef   Google Scholar

    [36]

    Rehman A, Weng J, Li P, Yu J, ur Rahman S, et al. 2023. Differential response of two contrasting melon (Cucumis melo L.) genotypes to drought stress. Journal of Plant Biology 66:519−34

    doi: 10.1007/s12374-023-09398-1

    CrossRef   Google Scholar

    [37]

    Amer MW, Awwad AM. 2021. Green synthesis of copper nanoparticles by Citrus limon fruits extract, characterization and antibacterial activity. Chemistry International 7:1−8

    doi: 10.31219/osf.io/76k8r

    CrossRef   Google Scholar

    [38]

    Iliger KS, Sofi TA, Bhat NA, Ahanger FA, Sekhar JC, et al. 2021. Copper nanoparticles: green synthesis and managing fruit rot disease of chilli caused by Colletotrichum capsici. Saudi Journal of Biological Sciences 28:1477−86

    doi: 10.1016/j.sjbs.2020.12.003

    CrossRef   Google Scholar

    [39]

    Zubair MS, Munis MFH, Alsudays IM, Alamer KH, Haroon U, et al. 2022. First report of fruit rot of cherry and its control using Fe2O3 nanoparticles synthesized in Calotropis procera. Molecules 27:4461

    doi: 10.3390/molecules27144461

    CrossRef   Google Scholar

    [40]

    Van Viet P, Nguyen HT, Cao TM, Van Hieu L. 2016. Fusarium antifungal activities of copper nanoparticles synthesized by a chemical reduction method. Journal of Nanomaterials 2016:1957612

    doi: 10.1155/2016/1957612

    CrossRef   Google Scholar

    [41]

    Batool F, Iqbal MS, Khan SUD, Khan J, Ahmed B, et al. 2021. Biologically synthesized iron nanoparticles (FeNPs) from Phoenix dactylifera have anti-bacterial activities. Scientific Reports 11:22132

    doi: 10.1038/s41598-021-01374-4

    CrossRef   Google Scholar

    [42]

    Kavitha KS, Baker S, Rakshith D, Kavitha HU, Yashwantha Rao HC, et al. 2013. Plants as green source towards synthesis of nanoparticles. International Research Journal of Biological Sciences 2:66−76

    Google Scholar

    [43]

    Sivakumar SR, Manimaran K, Govindasamy M, Alzahrani FM, Alsaiari NS. 2023. Green synthesis and characterization of CuO nanoparticles using Halymenia dilatata extract and its evaluation of antimicrobial, anticancer activity. Biomass Conversion and Biorefinery

    doi: 10.1007/s13399-022-03678-5

    CrossRef   Google Scholar

    [44]

    Veisi H, Karmakar B, Tamoradi T, Hemmati S, Hekmati M, et al. 2021. Biosynthesis of CuO nanoparticles using aqueous extract of herbal tea (Stachys Lavandulifolia) flowers and evaluation of its catalytic activity. Scientific Reports 11:1983

    doi: 10.1038/s41598-020-79139-8

    CrossRef   Google Scholar

    [45]

    Donga S, Chanda S. 2021. Facile green synthesis of silver nanoparticles using Mangifera indica seed aqueous extract and its antimicrobial, antioxidant and cytotoxic potential (3-in-1 system). Artificial Cells, Nanomedicine, and Biotechnology 49:292−302

    doi: 10.1080/21691401.2021.1899193

    CrossRef   Google Scholar

    [46]

    Thiruvengadam M, Chung IM, Gomathi T, Ansari MA, Gopiesh Khanna V, et al. 2019. Synthesis, characterization and pharmacological potential of green synthesized copper nanoparticles. Bioprocess and Biosystems Engineering 42:1769−77

    doi: 10.1007/s00449-019-02173-y

    CrossRef   Google Scholar

    [47]

    Nagar N, Devra V. 2018. Green synthesis and characterization of copper nanoparticles using Azadirachta indica leaves. Materials Chemistry and Physics 213:44−51

    doi: 10.1016/j.matchemphys.2018.04.007

    CrossRef   Google Scholar

    [48]

    Długosz O, Chwastowski J, Banach M. 2020. Hawthorn berries extract for the green synthesis of copper and silver nanoparticles. Chemical Papers 74:239−52

    doi: 10.1007/s11696-019-00873-z

    CrossRef   Google Scholar

    [49]

    Sadek ME, Shabana YM, Sayed-Ahmed K, Abou Tabl AH. 2022. Antifungal activities of sulfur and copper nanoparticles against cucumber postharvest diseases caused by Botrytis cinerea and Sclerotinia sclerotiorum. Journal of Fungi 8:412

    doi: 10.3390/jof8040412

    CrossRef   Google Scholar

    [50]

    Huang W, Fang H, Zhang S, Yu H. 2021. Optimised green synthesis of copper oxide nanoparticles and their antifungal activity. Micro & Nano Letters 16:374−80

    doi: 10.1049/mna2.12060

    CrossRef   Google Scholar

    [51]

    Mali SC, Dhaka A, Githala CK, Trivedi R. 2020. Green synthesis of copper nanoparticles using Celastrus paniculatus Willd. leaf extract and their photocatalytic and antifungal properties. Biotechnology Reports 27:e00518

    doi: 10.1016/j.btre.2020.e00518

    CrossRef   Google Scholar

    [52]

    Pariona N, Mtz-Enriquez AI, Sánchez-Rangel D, Carrión G, Paraguay-Delgado F, Rosas-Saito G. 2019. Green-synthesized copper nanoparticles as a potential antifungal against plant pathogens. RSC Advances 9:18835−43

    doi: 10.1039/C9RA03110C

    CrossRef   Google Scholar

    [53]

    Gomaa EZ, Housseiny MM, Ali Abdel-Khalik Omran A. 2019. Fungicidal efficiency of silver and copper nanoparticles produced by Pseudomonas fluorescens ATCC 17397 against four Aspergillus Species: a molecular study. Journal of Cluster Science 30:181−96

    doi: 10.1007/s10876-018-1474-3

    CrossRef   Google Scholar

    [54]

    Agarwal N, Solanki VS, Pare B, Singh N, Jonnalagadda SB. 2023. Current trends in nanocatalysis for green chemistry and its applications-a mini-review. Current Opinion in Green and Sustainable Chemistry 41:100788

    doi: 10.1016/j.cogsc.2023.100788

    CrossRef   Google Scholar

    [55]

    Angrasan JM, Subbaiya R. 2014. Biosynthesis of copper nanoparticles by Vitis vinifera leaf aqueous extract and its antibacterial activity. International Journal of Current Microbiology and Applied Sciences 3:768−74

    Google Scholar

    [56]

    Caroling G, Vinodhini E, Ranjitham AM, Shanthi P. 2015. Biosynthesis of copper nanoparticles using aqueous Phyllanthus embilica (Gooseberry) extract-characterisation and study of antimicrobial effects. International Journal of Nanomaterials and Chemistry 1:53−63

    Google Scholar

    [57]

    Tahir A, Quispe C, Herrera-Bravo J, Iqbal H, ul Haq Z, et al. 2022. Green synthesis, characterization and antibacterial, antifungal, larvicidal and anti-termite activities of copper nanoparticles derived from Grewia asiatica L. Bulletin of the National Research Centre 46:188

    doi: 10.1186/s42269-022-00877-y

    CrossRef   Google Scholar

    [58]

    Akintelu SA, Folorunso AS, Folorunso FA, Oyebamiji AK. 2020. Green synthesis of copper oxide nanoparticles for biomedical application and environmental remediation. Heliyon 6:E04508

    doi: 10.1016/j.heliyon.2020.e04508

    CrossRef   Google Scholar

    [59]

    Abd-Elsalam KA, Vasil'kov AY, Said-Galiev EE, Rubina MS, Khokhlov AR, et al. 2018. Bimetallic blends and chitosan nanocomposites: novel antifungal agents against cotton seedling damping-off. European Journal of Plant Pathology 151:57−72

    doi: 10.1007/s10658-017-1349-8

    CrossRef   Google Scholar

    [60]

    Sardar M, Ahmed W, Al Ayoubi S, Nisa S, Bibi Y, et al. 2022. Fungicidal synergistic effect of biogenically synthesized zinc oxide and copper oxide nanoparticles against Alternaria citri causing citrus black rot disease. Saudi Journal of Biological Sciences 29:88−95

    doi: 10.1016/j.sjbs.2021.08.067

    CrossRef   Google Scholar

    [61]

    Liu Z, Ren Z, Zhang J, Chuang CC, Kandaswamy E, et al. 2018. Role of ROS and nutritional antioxidants in human diseases. Frontiers in Physiology 9:477

    doi: 10.3389/fphys.2018.00477

    CrossRef   Google Scholar

    [62]

    Das S, Singh PK, Ameeruddin S, Kumar Bindhani B, Obaidullah WJ, et al. 2023. Ethnomedicinal values of Boerhaavia diffusa L. as a panacea against multiple human ailments: a state of art review. Frontiers in Chemistry 11:1297300

    doi: 10.3389/fchem.2023.1297300

    CrossRef   Google Scholar

    [63]

    Ssekatawa K, Byarugaba DK, Angwe MK, Wampande EM, Ejobi F, et al. 2022. Phyto-mediated copper oxide nanoparticles for antibacterial, antioxidant and photocatalytic performances. Frontiers in Bioengineering and Biotechnology 10:820218

    doi: 10.3389/fbioe.2022.820218

    CrossRef   Google Scholar

    [64]

    Emamverdian A, Ding Y, Barker J, Liu G, Hasanuzzaman M, et al. 2022. Co-application of 24-epibrassinolide and titanium oxide nanoparticles promotes Pleioblastus pygmaeus plant tolerance to Cu and Cd toxicity by increasing antioxidant activity and photosynthetic capacity and reducing heavy metal accumulation and translocation. Antioxidants 11:451

    doi: 10.3390/antiox11030451

    CrossRef   Google Scholar

    [65]

    Rajeshkumar S, Nandhini NT, Manjunath K, Sivaperumal P, Prasad GK, et al. 2021. Environment friendly synthesis copper oxide nanoparticles and its antioxidant, antibacterial activities using Seaweed (Sargassum longifolium) extract. Journal of Molecular Structure 1242:130724

    doi: 10.1016/j.molstruc.2021.130724

    CrossRef   Google Scholar

    [66]

    Djamila B, Eddine LS, Abderrhmane B, Nassiba A, Barhoum A. 2022. In vitro antioxidant activities of copper mixed oxide (CuO/Cu2O) nanoparticles produced from the leaves of Phoenix dactylifera L. Biomass Conversion and Biorefinery1−14

    Google Scholar

    [67]

    Saratale RG, Karuppusamy I, Saratale GD, Pugazhendhi A, Kumar G, et al. 2018. A comprehensive review on green nanomaterials using biological systems: recent perception and their future applications. Colloids and Surfaces B: Biointerfaces 170:20−35

    doi: 10.1016/j.colsurfb.2018.05.045

    CrossRef   Google Scholar

  • Cite this article

    Shah IH, Sabir IA, Ashraf M, Rehman A, Ahmad Z, et al. 2024. Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit. Fruit Research 4: e022 doi: 10.48130/frures-0024-0015
    Shah IH, Sabir IA, Ashraf M, Rehman A, Ahmad Z, et al. 2024. Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit. Fruit Research 4: e022 doi: 10.48130/frures-0024-0015
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Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit

  • 1.

    Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

  • 2.

    College of Horticulture, South China Agricultural University, Guangzhou 510642, China

  • 3.

    Bamboo Research Institute, Nanjing Forestry University, Nanjing 210037, China

  • 4.

    Department of Agriculture, University of Agriculture, Faisalabad 38000, Pakistan

  • 5.

    New Uzbekistan University, Mustaqillik Ave. 54, Tashkent 100007, Uzbekistan

  • 6.

    Anhui Dabieshan Academy of Traditional Chinese Medicine, Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resources, Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine, College of Biological and Pharmaceutical Engineering, West Anhui University, Lu'an 237012, China

  • 7.

    Biology Department, College of Science, King Khalid University, Abha 61413, Saudi Arabia

  • 8.

    Botany Section, School of Sciences, Maulana Azad National Urdu University, Hyderabad 500032, India

  • 9.

    School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

  • 10.

    Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW 2751, Australia

  • Received: 08 November 2023
  • Revised: 01 March 2024
  • Accepted: 12 March 2024
  • Published online: 03 June 2024
Fruit Research  4,  Article number: e022  (2024)  |  Cite this article

Abstract: Nanotechnology has emerged as a prominent field in recent times. The fabrication of biocompatible materials has taken on highlighted significance owing to their requisite application in diverse sectors including medicine, water treatment and purification, health, and other related fields. There has been a lot of research done recently on the green synthesis of various nanoparticles (NPs). Copper a high-performance metal used in agriculture to combat pathogenic attacks, has received less attention. The current work demonstrates the successful preparation of green synthesized copper oxide nanoparticles (CuO.NPs) from Mangifera indica (M. indica) leaf extract. The spectral and morphological characterization biosynthesized were observed using, FTIR, XRD, and TEM analysis. The FTIR analysis revealed the functional groups present in plant extracts. XRD was carried out to demonstrate the crystalline nature and size of nanoparticles using the Scherrer formula. UV was performed to observe the optical properties of NPs. Further, Transmission electron spectroscopy (TEM) was carried out to confirm the physical shape of CuO.NPs with 50 nm. The M. indica mediated NPs were evaluated against gram-negative and positive bacteria Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) at different concentrations. The in vivo fungicidal activity was performed against Rhizophus oryzae (R. oryzae) on Diospyros kaki (persimmon fruit). The detach fruit method was applied to evaluate the potential of NPs. Higher exposure of 100 µg·mL−1 CuO.NPs showed mycelia inhibition followed by 30, 60, and control treatments. Furthermore, Green CuO.NPs showed prominent antioxidant activities as compared to plant sources. The findings obtained suggest that the green-formulated CuO.NPs could be further investigated for the treatment of many phytopathogenic diseases.

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    Introduction

    • A sustainable increase in agricultural production is needed due to a predicted increase of 100%−110% in demand for crops globally from the year 2005 to 2050[1]. Abiotic stresses can physically damage the plant or weaken the plant's defense and health, thus assisting the entry or attack of pathogens on the plant[2]. Biotic stresses mainly involve infections caused by pathogens, such as viruses, fungi, nematodes, bacteria, and protozoa[3]. Disease in plants is defined as the response of plant cells and tissues to any environmental stress or a pathogen, resulting in abnormality in plant health[4]. Disease endemics in plants occur yearly on various crops in different parts of the world[5]. Phytopathogens have a severe negative impact on the quality and quantity of agricultural products, parallelly a bad effect on the economy of a country. Thus, a sustainable food supply is at risk, due to the rapid propagation of pathogens with an increase in the incidences and severity of infectious diseases[6].

      At present, the increasing demand for crop production depends on synthetic agrochemicals to reduce the risks posed by plant diseases and ensure crop yield[7]. The Food and Agriculture Organization of the United Nations (FAO) Statistics database shows that the use of fungicides, bactericides, and pesticides worldwide is increasing rapidly[8]. However, excessive use of agrochemicals without discrimination contributes to global warming and has several adverse effects—such as making pathogens more resistant, causing toxicity to non-target organisms, and posing severe risks to human and environmental health[9,10]. Consequently, innovative plant disease control approaches and advancements in technology need to be established and employed to enhance the effectiveness of plant disease control measurements and minimize environmental damage. Nowadays, antimicrobial nanomaterials are attracting more attention in the scientific community as they can unlock the limitations experienced by other antimicrobial agents and traditional pesticides. Nanomedicine is a new approach to overcoming the challenges of conventional treatments, based on the manufacturing and fabrication of nanoparticulate[11,12]. Numerous types of nanostructures such as metal nanoparticles, nanogels, biodegradable polymeric nanoparticles, nanoliposomes, and solid lipid nanoparticles, have been endeavored as probable drug delivery systems[13,14]. Nanoparticles are generally no larger than 100 nm in size and their smaller size along with their higher surface-to-volume ratio, govern them, as effective biocidal agents, as both these combined effects facilitate the intimate interaction on the microbial membrane[13,15]. The fungicidal ability of biosynthesized metallic nanoparticles was found to be more aggressive than commercially applied antibiotics such as amphotericin and fluconazole. Obvious membrane damage in Candida sp. has been observed after the application of plant-mediated silver nanoparticles, which not only damaged the intercellular components of the fungus but also destroyed the cell functions[16,17]. In antibacterial activity, the negative charge on the bacterial cell wall interacts with the positive charge nanoparticles due to their electrostatic forces resulting in the disruption of the bacterial cell walls. Furthermore, nanoparticles also release metal ions from their extracellular place, which then enter the cell wall and disrupt the normal biological process of bacteria. Inside the bacterial cell, either nanoparticles or metal ions, persuade the ROS level and damage the protoplasm. The generation of oxidative stress leads to the oxidation of glutathione, which results in the destruction of the antioxidative defense mechanism of organisms against ROS. The metal ions are therefore free to interact with cellular systems (e.g., membranes DNA, proteins, etc.), disrupting cellular functions. Metal ions can develop a solid coordination bond with O, S, or N atoms which are present in abundance in biomolecules and organic systems. Meanwhile, the bond between biomolecules and metal ions is generally non-specific, ultimately, metallic nanoparticles exhibit a broad spectrum of potential[18,19].

      To synthesize eco-friendly and low-cost metallic nanoparticles, researchers are utilizing the capabilities of biological materials to manufacture metallic nanoparticles[20]. In the green-fabrication of metallic nanoparticles, the reduction of metals involves the biological mass as reducers—either intra-cellularly or extra-cellularly[21]. Apart from eco-friendliness and cost-effectiveness, the advantage of the biological approaches over classical approaches (physical and chemical methods), comprise the efficiency of the technique in catalyzing the reactions in the aqueous environment at standard pressure and temperature conditions, as well as the flexibility of the process by implemented in almost at any scale and any settings[22]. The elements of biological sources are responsible for the reduction and the process may often be triggered by various compounds and constituents such as proteins, terpenoids, flavonoids, alkaloids, terpenoids, phenols, amines, carbonyl, amide groups, different pigments, and other reducing agents. One or more of these reducing agents may be responsible for the synthesis of metallic nanoparticles[23]. Applications of nanomaterials are extended to the fields of both human and plant pathology and biotechnology[24,25]. Based on their unique characteristics, various biogenically synthesized nanoparticles serve as anticancer and antimicrobial agents in various fields[26,27]. CuO nanoparticles have vast unique characteristics from pharmacology to pest ecology for overcoming various pathogenic diseases[28].

      Mangifera indica is a famous tree that belongs to the family Anacardiaceae used in traditional medicines, especially for skin problems, cough, diarrhea, malaria, dysentery jaundice, and their anti-microbial properties[29,30]. Thus, due to the antioxidant, antimicrobial, and pharmacological importance of M. indica, it was assumed that employing plant leaves as a reducing and capping agent would enable the formation of CuO.NPs with vital phytochemicals. The main objective of the current study is to synthesize eco-friendly and economically viable M. indica-mediated CuO.NPs to explore their antifungal and antibacterial potential. This current work shows that CuO.NPs synthesized by M. indica are biocompatible, eco-friendly, and environmentally sustainable Nano fungicides against phytopathogens. Therefore, improving the yield of nanoscale metal particles, using low-cost raw materials, and greensource, and employing simple energy-saving technology are the research directions needed in the future. At present, there have been successful cases of using grass to synthesize CuO.NPs. Therefore, green synthesis of nanoscale metals may have a broad prospect and a great potential for development.

    Materials and methods

      Chemicals and pathogenic strains

    • The chemicals used in this work were Copper (II) sulphate pentahydrate (CuSo4·5H2O), deionized H2O. Escherichia coli, Staphylococcus aureus and Rhizopus oryzae.

      Plant material processing

    • The healthy leaves of Mangifera indica were collected from Punjab province, Pakistan, and identified from the well-reputed plant herbarium center of the taxonomy laboratory department at Quaid-i-Azam University (QAU) Islamabad Pakistan. The fresh plant leaves were thoroughly washed with de-ionized water and dried under ambient temperature (1−2 weeks). After plant materials had completely air dried, extracts were pulverized with a blender to a fine powder. Afterward, 30 g of plant powder was added to 300 mL of distilled water and boiled for half an hour at 74 °C. The material was then shifted in an incubator shaker for 2−3 h at 50 rpm at 40 °C. The resultant mixture was then filtered with Whattman No. 1 filter paper. The extract filtered was kept at 4 °C for further synthesis process of CuO.NPs[31].

      Preparation of CuO.NPs

    • The formulation of green CuO.NPs were mediated by the co-precipitate method by applying Mangifera indica leaf extract. For the green synthesis of CuO.NPs, 40 mL copper (II) sulphate pentahydrate (CuSo4·5H2O) solution (1 M) was prepared by adding in 10 mL aqueous plant extract and stirring on a hot plate at 80 °C for 4 h at 200 rpm. The brownish color CuO.NPs in precipitate form were collected via 12,000 rpm of centrifugation (Velocity 14R, 220VAC 50/60HZ, 10A China), for 10 min. The resulting pellet was washed thrice with deionized water and dried in a drying oven for 4 h at 60 °C. Finally, the radish-brown NPs were collected, and stored at room temperature for further use[32].

      Phytochemicals screening

    • The antioxidant, antifungal, and antibacterial compounds in plant extracts were carried out by manual procedure in a lab using chemicals. The phytochemical screening of phenols and saponins, alkaloids triterpenes, flavonoids, and tannins according to the already well-established protocol[33].

      Phenols and tannins test verification

    • To confirm the presence of phenols and tannins in M. indica plant extract solution. A few drops of FeCl3 3% were added drop by drop to a 1 mL extract solution. After some time, deep blue coloration formed which was the first identification of phenolic and tannins.

      Test for triterpenes

    • For triterpene compound analysis, 1 mL of chloroform was added into the solutions, and then 1 mL of concentrated H2SO4 was carefully mixed into the test tubes by sliding it down the walls. The confirmation of triterpenes' presence was indicated by the development of red coloration in the solution.

      Test for flavonoids

    • Lead acetate solution was added to 1 mL of the extract solutions and the presence of flavonoids was confirmed by the formation of a yellow precipitate in the solution.

      Test for alkaloids

    • The alkaloid test was carried out by adding 5 mL of HCl to the extract solution. The solution was agitated, filtered, and kept for further analysis. Meyer's test was then carried out by adding 2 mL of the filtrate to 5 mL of Meyer's reagent. The formation of a yellow precipitate indicates the presence of alkaloids.

      Characterizations of CuO.NPs

    • The crystallographic nature and intrinsic characteristics of green synthesized M. indica-mediated NPs were confirmed by XRD analysis (Schimadzu-Model Kyoto, Japan) ranging 2θ from 10° to 90° via Cu/Kɑ radiations with wavelength 1.5406 Å ran at 40 kV with 30 mA at room temperature. The size of synthesized particles was determined using the Debye-Scherer equation (D = K/cos), where D is the crystal size and is vertical to the reflecting planes. K is constant in this equation (0.9), and the variables are the X-ray wavelength (1.5406), the angular full-width at half-maximum in radians, the Bragg's angle, and the angular full-width at half-maximum in radians.

      FT-IR analysis for the green synthesized M. indica-mediated CuO.NPs were analyzed to confirm the functional groups of biomolecules in the prepared NPs. The wavelength was set in the range of 400−4,000 cm−1 using an FT-IR spectroscope (Schimadzu-Model FT-IR, Kyoto, Japan) having a resolution of 4 cm−1.

      The surface morphology of green synthesized CuO.NPs were assessed by scanning electron microscopy. The drop of metal NP solution sample was loaded on copper-coated stubs and then evaporated at light for full drying. Finally sample was loaded in the transmission electron microscope (JEOL JSM-5910), with 10 KV of accelerating voltage for 4 min[34]. The morphology of synthesized samples was further proven with a high-resolution application of the protocol. The size of the synthesized particles was measured by determining the diameter of the nanoparticles[35,36].

      Antibacterial assay

    • The antibacterial potential of synthesized green CuO.NPs were evaluated against E. coli and S. aureus bacteria via the disc diffusion method[37]. The bacterial inoculum was prepared by culturing a single colony of E. coli in nutrient broth at 37 °C for 24 h. Then the prepared fresh inoculum was swabbed evenly on media plates (nutrient broth agar), and 5 mm of wells were created in the plates by using an autoclaved cork borer. A total volume of 20 µL of the sample having various concentrations (100, 50, and 30 µg·mL−1) was added according to the wells. The cefixime was applied as a positive control and DMSO as a negative control. The culture plates were incubated at 37 °C for 24 h and their zones of inhibitions were measured. The experiment was performed three times.

      Antifungal assay

    • The antifungal potential of synthesized green CuO.NPs were explored in vivo against R. oryzae. In vivo was accomplished by adding R. oryzae mycelia in Czapek Dox Broth medium. The components of Czapek Dox Broth media were sucrose (7.5 g), potassium chloride (0.25 g), sodium nitrate (0.5 g), magnesium sulphate (0.25 g), dipotassium phosphate (0.25 g), ferrous sulphate (0.005 g), and 250 mL distilled H2O. The media was incubated at 32 ± 2 °C for 24 h at 50 rpm with continuous shaking. The D. kaki was surface sterilized via 50 % sodium hypochlorite suspension before treatment with synthesized CuO.NPs. Then the artificial wounds were created in the center of the fruits by a 2 mm sterile cork borer. In the wound of fruits, 5 µL spore suspension was inoculated into the wounds of each fruit, further treated with 30 µL volume of both concentrations (100 and 60 µg·mL−1) of CuO.NPs. To provide moisture and avoid contamination, the treated fruits were covered with a wet muslin cloth. After a week of post-inoculation, the diameter of the lesion was measured[38,39].

      Antioxidant activity

    • The antioxidant activity was assessed using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging experiment, 100 μL of green synthesized CuO.NPs at a concentration of 1 mg·mL−1 were serially diluted with 100 μL of methanol. The well was filled with 100 μL of 0.1 mM methanolic DPPH, and the plates were then let to sit at room temperature in the dark for 30 min. M. indica extract, CuO.NPs, and ascorbic acid concentrations were evaluated at an ELISA plate reader were used to read the plates at 517 nm. Where A0 and A1 are the absorbances of the control and treatment, respectively, were used to calculate the DPPH inhibitory percentage[34].

      DPPHinhibitory%=A0−A1A0×100

      Where A0 and A1 are absorbances of control and treated, respectively.

    Results and discussion

    • In the last few years, nanotechnology has attained great impetus in applied material due to its unique physiochemical properties. Numerous methods for synthesizing metal nanoparticles are currently documented in the literature and employed by researchers in their quest to discover novel and emerging uses for these nanoparticles. Nanoparticles are the upcoming plant pathogen weapons in modern agriculture, and green-produced nanoparticles have shown significant potential for disease prevention[40]. The biological approach encompasses the creation of nanoparticles through the utilization of living organisms, including plants, bacteria, fungi, and algae. When plants are involved in the synthesis of nanoparticles, it is referred to as green nanotechnology[41]. The utilization of plant biomaterials for the synthesis of metallic nanoparticles is now recognized as a highly emerging field. Plants possess inherent organic reducing agents, which render them well-suited and adaptable for nanoparticle synthesis[42]. Numerous plant extracts have been effectively tested for the production of metallic nanoparticles. Extracts from various plants such as Camellia sinensis, Azadirachta indica, Halymenia dilatata, Stachys lavandilfolia, Eucalyptus, and Mentha have been documented as capable of synthesizing diverse types of CuO.NPs, each with distinct and specific applications[38,43,44].

      Mangifera indica, a significant plant with numerous medicinal uses, has gained recognition. Phoenix dactylifera has previously been employed for the production of various metallic nanoparticles. To investigate the antimicrobial and antioxidant activity, researchers have utilized leaf extract from M. indica to synthesize silver oxide nanoparticles (AgNPs)[45]. In the present study, M. indica was utilized as a source to generate copper oxide nanoparticles (CuO.NPs). The synthesis of green M. indica-mediated CuO.NPs were confirmed via a color change from yellow to copper-reddish indicating CuO.NPs synthesis. The main objective of green synthesis was the fabrication of cost-effective, environmentally friendly, and biocompatible nanoparticles[46]. The production of green CuO.NPs are a complicated process that depends on various variables due to their colloidal properties. The main objective is to develop a phyto-fabricated approach that can be commercialized without using complex processes such as magnetic filtration, ultracentrifugation, co-precipitation, and flow field gradient. Another objective is to specify the reaction conditions that aid in controlling the size of the particle. Already reported studies confirmed the suitability of the co-precipitation method to synthesize reduced size and homogenous texture. The results of this study align with previous research, demonstrating that this method can meet the increasing need for well-dispersed green CuO.NPs in biomedicine and engineering[47]. In this present work, green synthesized CuO.NPs were characterized through different techniques. After visual confirmation of biomolecules from M. indica plant extract. To confirm the antifungal and antibacterial biological molecules in green source plant extract visual confirmatory tests were performed in the laboratory. The Mangifera indica filtrate solution contained all antioxidant flavonoids necessary for the reduction of green synthesized CuO.NPs (Fig.1).

      Figure 1. 

      Proposed hypothetical picture of the reduction mechanism of copper sulphate by the aqueous leaf extract solution of M. indica on a hot plate at 80 °C for 4 h at 200 rpm.

      FTIR spectroscopy was carried out to investigate biomolecules responsible for the reduction of Cu nanoparticles. The functional groups act as capping and reducing agents in synthesizing nanoparticles and were analyzed by FT-IR (Fig. 2). FT-IR exhibited absorption peaks 3,404.13, 300.37, 2,382.35, 1,641.84, 1,476.90, 1,072.42, 872.66, and 651.51 cm−1 matching to several functional groups that are C-N stretching aromatic amino groups, C-O carboxylic anions, alcohol O-H stretching, and amine N-H stretching groups respectively, while peaks for CuO.NPs were instituted nearby 550–600 cm−1. The fabricated CuO.NPs were covered by proteins and various metabolites occupying functional groups. The FT-IR result confirmed that the proteins and amino acids residues built a strong connection to attract the metals and prevent clusters due to the capping of CuO.NPs and stabilizing the respective medium (Table 1).

      Figure 2. 

      FTIR spectra of M. indica mediated CuO.NPs. FTIR showed corresponding peaks 3,404.13, 300.37, 2,382.35, 1,641.84, 1,476.90, 1,072.42, 872.66, and 651.51cm−1 corresponding to various functional groups.

      Table 1.  Possible functional groups in M. indica plant extract.

      S.
      No.      		Functional groupsCompoundsWave.
      no.      		VibrationBondingPeaks
      1OHAlcohol3,404.13StretchingStrongBroadband
      2O-HCarboxylic300.37StretchingStrongBroadband
      3O=C=OCarbon oxide2,382.35StretchingMediumBroadband
      4−C=CAlkanes1,641.84StretchingMediumBroadband
      5N-HAmine1,476.90BendingMediumBroadband
      6N-HAnhydride1,072.42StretchingStrongBroadband
      7C-ClAldehyde872.66BendingMediumBroadband
      9C-BrAlkyl Halides651.51StretchingStrongBroadband

      The current FT-IR analysis demonstrates that green synthesized CuO.NPs have potential as an organic core through the presence of various functional groups, as well as an inorganic support to penetrate the cell wall of microorganisms. Previous studies have indicated that green synthesized CuO.NPs have a variety of reactive functional groups, providing enough potential to be utilized as an effective means of combating pathogens in the agricultural sector[48]. The FT-IR analysis verified the existence of functional groups in the green synthesized CuO.NPs, which possess the potential to effectively inhibit pathogenic growth.

      The XRD spectra of synthesized green CuO.NPs demonstrated six major peaks at 2θ angles of 49.67, 39.36, 36.75, 29.56, and 25.34 corresponding to 113, 202, 111, 002, −111, and 110 plans respectively depicting the crystalline nature of nanoparticles (Fig. 3). However, a few unknown peaks were also noticed denoted by *. The average size of the particles was evaluated by Scherrer's equation (D = kλ/β Cosθ) and was retrieved from XRD findings to be 55.29 nm. The XRD results are compatible with the previously studied XRD patterns[49]. The XRD patterns portray the crystalline texture of synthesized particles which play an important role in the interaction of nanoparticles with microbial cell walls[50]. Moreover, the crystalline nature of small-size particles helps to control the formation of biofilm by producing oxidative stress[38].

      Figure 3. 

      XRD spectra of M. indica mediated CuO.NPs. The XRD spectra of the green synthesized M. indica mediated CuO.NPs exhibit seven main peaks at 2θ angles of 49.67, 39.36, 36.75, 29.56, and 25.34 plains respectively.

      The TEM analysis indicated that the CuO.NPs display a spherical morphology and are effectively dispersed without aggregating, with sizes ranging from 40−80 nm, corresponding closely to the sizes determined from the XRD analysis (Fig. 4a & b). The current analysis is in correspondence with the already reported analysis of green fabricated CuO.NPs. The particle aggregation can be attributed to the electrostatic attraction forces between them. Previous studies suggest that a spherical object can easily enter the microbial cell wall which is mainly responsible for sustaining the integrity of microbes and disturbances in cell walls eventually leading to microbial death[51,52].

      Figure 4. 

      TEM analysis of green synthesized M. indica CuO.NPs. Spherical shape with a diameter ranging between 30−90 nm.

      The TEM results of CuO.NPs shown in Fig. 4 demonstrated a spherical shape having a diameter in the range of 30−90 nm. The electrostatic forces among the particles result in slight agglomeration, but the presence of organic core and plant extract material provides a clear demarcation for each particle (Fig. 4). The shape is likely to increase the surface area of both the particles and the substrate, thereby enhancing their ability to attach and ultimately leading to the antimicrobial effects of CuO.NPs[53].

      Antimicrobial activities of CuO.NPS

    • The antibacterial activity of CuO.NPs were evaluated at different doses (30, 60, and 100 µg·mL−1) against E. coli and S. aureus (Fig. 5). In E. coli, the results showed the highest inhibition antibacterial zone at 100 µg·mL−1 of CuO.NPs at 10.8 ± 0.41 (mean ± SD) while 60 and 30 µg·mL−1 from three repeated trials showed inhibitions respectively relative to control 6.4 ± 0.20 and 3.6 ± 0.20 (mean ± SD). Similarly, it was found that green CuO.NPs inhibited the growth of gram-positive bacterial stain efficiently 8.89 ± 0.69 at 100 µg·mL−1 compared with other treatments. While 60 µg·mL−1 of NPs showed a 6.0 ± 0.2 mm zone of inhibition. In the current findings, 3.8 ± 0.2 inhibition zone was observed at the lowest concentration of 30 µg·mL−1 of CuO.NPs. Previously reported studies showed that the bactericidal effects of green fabricated NPs are due to the small size of particles and the availability of various organic functional groups on the nanoparticle's surface[54]. It was reported in a previous study, CuO.NPs found to have highly bactericidal potential to stop their growth. The organic base of nanoparticles attracts the bacterial cells due to strong electrostatic forces of attraction, resulting in the deactivation of the cellular enzymes and causing damage to plasma membrane permeability ultimately causing cell death[55,56]. Moreover, the accumulation of different phytocompounds on the surface of CuO nanoparticles causes damage to the microbial DNA and their proteins formulating enzymes and inhibiting microbial growth. Additionally, the particles' small sizes and crystalline texture were verified by XRD, SEM, and TEM causing the inhibitory effects of the CuO.NPs[57].

      Figure 5. 

      In-vitro antibacterial activity of CuO.NPs were evaluated at different doses (30, 60, and 100 µg·mL−1) against E. coli and S. aureus.

      The antifungal activity in vivo of green synthesized CuO.NPs were evaluated on detached fruit assay. The in vivo study on persimmon fruit resulted in an obvious reduction of disease with an increase in the doses of synthesized CuO.NPs (100, 60, and 30 µg·mL−1) (Fig. 6). In vitro, the highest concentration of 100 µg·mL−1 of particles showed maximum inhibition of disease incidence 23.4 ± 1.81 (mean ± SD) against R. oryzae as compared to control 97.0 ± 0.81 (mean ± SD) (Table 2).

      Figure 6. 

      Fruit detached antifungal in vivo assay. Persimmon fruits were infested and treated with different concentrations of CuO.NPs including: (a) 30 mg·L−1, (b) 60 mg·L−1, (c) and (d) 100 mg·L−1. Control fruit were without any NP.

      Table 2.  Effectiveness of green CuO.NPs on fruit against disease.

      Treatment Diseased area (mm)
      30 mg·L−164.6 ± 1.6
      60 mg·L−144.2 ± 0.6
      100 mg·L−123.4 ± 1.8
      Control97.0 ± 0.81

      In this current work, antifungal potential has been shown by green fabricated CuO.NPs both in vivo and in vitro. The presence of plant phytocompounds on the surface of nanoparticles is the main reason for their potential and the ability of CuO.NPs to penetrate fungal cell walls are also dependent on their small size and crystalline texture. The small size of the particles provides a large surface area to adsorb the biomolecules to the cell wall and results in disruption of the cell wall and cellular components, leading to the death of microorganisms. The green fabricated CuO.NPs exhibit potential microbicidal effects, cost-effectiveness, and reproducibility over chemically generated nanoparticles[58]. To identify synergistic effects, copper nanocomposites were tested against the phytopathogenic fungus Alternaria alternata, Rhizoctonia solani, and Botrytis cinerea. According to the findings, nanocomposite showed greater activity at a concentration of 90 g·mL−1. Bimetallic blends antifungal properties were similarly successful in controlling Rhizoctonia solani growth at doses of 30, 60, and 90 g·mL−1. In a greenhouse setting, they also demonstrated successful management of cotton seedling damping-off[59,60].

      Antioxidant activity

    • In humans, free radicals are created by several metabolic pathways and cause degenerative diseases and lowered immune function (Liu et al.)[61]. To combat free radicals, medicinal plants with phenolic components are employed as antioxidants. The proportion of scavenging action was substantially higher in the standard and plant extracts, Das et al.[62] reported comparable findings. While Mangifera indica contain polyphenols and other phytochemicals with significant antioxidant activity and are employed as natural antioxidants to control degenerative diseases (Ssekatawa et al.)[63], ascorbic acid is a recognized antioxidant. The mechanism behind the antioxidant activity of inorganic nanoparticles involves the binding of transition metal ion catalysts by free radicals, which results in the enhancement of radical scavenging activity. Antioxidant activity was noticeably elevated in the green synthesized NPs and ascorbic acid. The capping of the CuO.NPs with the phytochemicals utilized in their production were confirmed by FTIR analysis. The biosynthesized CuO.NPs antioxidant activity against the DPPH radical was assessed, and its efficacy was plant extract and conventional ascorbic acid. CuO.NPs had a potent inhibitory effect on DPPH, with an IC50 value of 10.68 0.03 g·mL−1. A dose-dependent reaction of CuO.NPs and ascorbic acid to the DPPH radical are shown in Fig. 7.

      Figure 7. 

      Antioxidant activity of M. indica CuO.NPs. The DPPH assay was carried out with 20, 40, 80, 160, and 320 µg·mL−1 concentrations of M.indica. CuO.NPs and Ascorbic acid were used as a standard.

      CuO.NPs biosynthesized with Sargassum longifolium demonstrated an inhibitory percentage of 20% when tested at 5 g·mL−1, which is consistent with our previous findings[64,65]. On the other hand, copper mixed oxide (CuO/Cu2O) nanoparticles made from Phoenix dactylifera leaves' antioxidant properties showed strong DPPH inhibition at 4 mM[66]. Green chemistry of inorganic metal and metal oxide nanoparticles (NPs) have a wide range of applications in agriculture, particularly in the management and treatment of plant diseases due to fungi. For the correct uses, the biocompatibility and toxicity of the nanomaterials should be investigated[67].

      Nanoparticles exhibit antimicrobial and antioxidant potential through multifaceted penetration mechanisms. The nanoparticles bind to microbial membrane and their penetration inside the protoplasm of pathogenic cells has been documented as the most prominent mode of antimicrobial action. NPS released into the microbial cells react with thiol groups causing a denaturation of the proteins and enzymes Fig. 8. The results demonstrated that green modified CuO.NPs can represent a successful alternative treatment for fungi and bacterial infections.

      Figure 8. 

      Expected antimicrobial mechanism of CuO.NPs. Disruption of cell wall and cytoplasmic membrane nanoparticles adhere to or pass through cell wall and cytoplasmic membrane. Cu ions denature ribosomes and inhibit protein synthesis. Interruption of adenosine triphosphate (ATP) production: ATP production is terminated because Cu ions deactivate respiratory enzymes on the cytoplasmic membrane. Reactive oxygen species (ROS) produced by the broken electron transport chain can cause membrane disruption. Cu and reactive oxygen species bind to deoxyribonucleic acid and prevent its replication and cell multiplication. CuO.NPs directly move across the cytoplasmic membrane, which can release organelles from the cell.

    Conclusions

    • The current work provides an efficient and cost-effective protocol for the synthesis of CuO nanoparticles by the green approach and its safe use in phytopathology. Using plant materials for particle synthesis allows for the safe and effective use of these particles as antimicrobial agents to combat diseases. This approach is essential in meeting the growing demand for biocompatible solutions in food and agriculture. In this current work, the nanoparticles used against pathogens suppressed their growth by damaging their cell walls and delaying their spore germination. Based on our findings, it is highly recommended to apply green synthesized CuO.NPs mitigate devastating and lethal plant diseases due to their significant antimicrobial effects. This work offers a new avenue for additional investigations of metallic nanoparticles in vital domains like phytopathology and agriculture. The use of this environmentally friendly method of biogenic nanoparticles may help to reduce the fungal infection in sweet fruit and improve the socio-economic standing of farmers.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception, literature analysis data and figures desigen: Shah IH, Ashraf M, Ashraf GA, Rasheed HU, Li G, Mouna J, Faizan M, Altaf MA, Shakoor A; experiments, writing, and prepared the original draft: Shah IH; reviewing data and table creation: Sabir IA, Azam M, Rehman A, Ahmad Z; providing guidance on the whole manuscript: Song C, Manzoor MA. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

      Acknowledgments

      • This work was supported by the Key R&D Project of the 14th Five Year Plan of China (2023YFC3503804), Startup fund for high-level talents of West Anhui University (WGKQ2022025), the Open Fund of Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine (WXZR202318), and Demonstration Experiment Training Center of Anhui Provincial Department of Education (2022sysx033). The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for funding this work through Small Groups Project under Grant Number (RGP.2/176/44). We thank Dr. Aamir Hasan Shah from the University of California Loss Angles USA for CuO.NPs characterization analysis of XRD and FTIR.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Iftikhar Hussain Shah, Irfan Ali Sabir

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      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Shah IH, Sabir IA, Ashraf M, Rehman A, Ahmad Z, et al. 2024. Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit. Fruit Research 4: e022 doi: 10.48130/frures-0024-0015
    Shah IH, Sabir IA, Ashraf M, Rehman A, Ahmad Z, et al. 2024. Phyto-fabrication of copper oxide nanoparticles (NPs) utilizing the green approach exhibits antioxidant, antimicrobial, and antifungal activity in Diospyros kaki fruit. Fruit Research 4: e022 doi: 10.48130/frures-0024-0015
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  • Introduction
  • Materials and methods

  • Chemicals and pathogenic strains

  • Plant material processing

  • Preparation of CuO.NPs

  • Phytochemicals screening

  • Phenols and tannins test verification
  • Test for triterpenes
  • Test for flavonoids
  • Test for alkaloids

  • Characterizations of CuO.NPs

  • Antibacterial assay

  • Antifungal assay

  • Antioxidant activity

  • Results and discussion

  • Antimicrobial activities of CuO.NPS

  • Antioxidant activity

  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
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