Application of Graphene Oxide Nanomaterials in Crop Plants and Forest Plants

Abstract

Graphene oxide (GO) is a carbon-based nanomaterial explored for agricultural and forestry uses, but plant responses are strongly subject to both the dose and the route of exposure. We summarized recent studies with defined graphene oxide (GO) exposures by seed priming, foliar delivery, and root or soil exposure, while comparing annual crops with woody forest plants. Mechanistic progress points to a shared physicochemical basis: surface oxygen groups and sheet geometry reshape water and ion microenvironments at the soil–seed and soil–rhizosphere interfaces, and many reported shifts in antioxidant enzymes and hormone pathways likely represent downstream stress responses. In crops, low-to-moderate doses most consistently improve germination, root architecture, and tolerance to salinity or drought stress, whereas high doses or prolonged root exposure can cause root surface coating, oxidative injury, and photosynthetic inhibition. In forest plants, evidence remains limited and often relies on seedlings or tissue culture. For forest plants with long life cycles, processes such as soil persistence, aging, and multi-seasonal carry-over become key factors, especially in nurseries and restoration substrates. The available data indicate predominant root retention with generally limited root-to-shoot translocation, so residues in edible and medicinal organs remain insufficiently quantified under realistic-use patterns. This review provides a scenario-based framework for crop- and forestry-specific safe-dose windows and proposes standardized endpoints for long-term fate and ecological risk assessment.

1. Introduction

With the continuous increase in the global population and the intensification of climate change, modern agriculture is under pressure to improve crop yield, quality, and stress resilience under constrained resources. Nanotechnology provides new technological pathways for the sustainable development of agriculture [1]. Typical nanomaterials have at least one dimension in the range from 1 to 100 nm and possess a high specific surface area and abundant surface-active sites. These features enable them to cross plant cell walls and other barriers and to act on specific plant tissues or cellular structures with relatively high precision, which offers the potential to enhance the use efficiency of agricultural inputs and to reduce environmental burdens.

In plant science and agricultural applications, research on nanomaterials has evolved from early work on biosensing and simple delivery to direct intervention in plant life processes [2]. On the one hand, nanomaterials can function as efficient carriers that deliver fertilizers, pesticides, or genetic cargo into plants in a targeted manner, thereby enabling controlled release and functional gene modulation. On the other hand, some nanomaterials can act as nanobiostimulants that regulate metabolism and antioxidant defense, and in this way enhance crop tolerance to stresses such as drought, salinity, and heavy metal exposure [3,4]. The specific biological effects of these materials depend strongly on their chemical composition, particle size, morphology, and surface chemistry.

Among various nanomaterials, carbon-based nanomaterials, particularly graphene and its derivatives, have attracted extensive attention [5,6]. Compared with highly hydrophobic graphene, graphene oxide (GO) carries abundant oxygen-containing functional groups on its basal planes and edges, which confer favorable hydrophilicity, colloidal stability, and ease of functionalization, making GO suitable for use in aqueous plant systems. In recent years, many studies have investigated the roles of GO in regulating seed germination, shaping root system architecture, and alleviating both abiotic and biotic stresses in plants [7]. These studies have also revealed pronounced dose-dependent and species-specific effects, with potential phytotoxicity and environmental risks at high doses. Against this background, this review synthesizes recent evidence to address three questions: (1) How do GO physicochemical traits control its environmental behavior and bioavailability at the plant–soil interface? (2) Which exposure routes, dose windows, and developmental stages drive beneficial responses versus phytotoxicity, and how do these patterns differ between annual crops and woody forest species? (3) Which risk factors, endpoints, and mitigation strategies are required to enable safe application? We further discuss emerging opportunities of GO in gene delivery, nano-enabled agrochemicals, and plant biosensing to support smart and sustainable agroforestry.

2. Physicochemical Basis of GO-Plant Interactions

GO is a graphene derivative enriched with oxygen-containing functional groups [8,9]. Hydrophilic surface groups, together with nanoscale capillary channels formed by sp2 carbon domains, enable GO to disperse readily in aqueous environments and soil pores and to exhibit strong water adsorption and transport capacity [10]. At the soil-to-seed interface, GO can adsorb and retain water within soil pores and on the seed coat, and it can facilitate directional water transport along nanosheets and capillary networks toward the seed surface and micropores of the seed coat, thereby improving water availability during germination and accelerating germination kinetics [11]. When GO is well dispersed across soil layers, it can attach to soil particles and the rhizosphere microenvironment, increase local water content, and slow water loss. This dual regulation pattern that combines local water retention with directional water transport provides a physicochemical explanation for growth-promoting effects observed at early germination stages in several studies [12]. During rhizosphere transport and potential entry into plant tissues, uniformly dispersed GO has also been reported to show limited aggregation on root surfaces, which may reduce physical blockage of water and nutrient uptake and partially explain the relatively low toxicity observed under some exposure conditions [13].

Beyond these general functions, GO should be viewed as a family of materials rather than a single fixed substance [10]. Key descriptors include oxidation degree, commonly reflected by the C-to-O ratio or oxygen content, the abundance and spatial distribution of oxygen-containing functional groups, defect density, sheet lateral size and thickness, and surface charge [9]. These physicochemical descriptors jointly determine acidity, adsorption capacity for nutrient ions and metals, and colloidal behavior in exposure media, and they can therefore drive inter-study variability even when nominal doses are similar. Comparative analyses of graphene-derived nanomaterials from a structure–function perspective suggest that GO can show stronger root-promoting effects than less-oxidized graphene forms in some species, which has been attributed to higher defect density, more abundant surface functional groups, and stronger negative surface charge [12,13]. Collectively, oxygen-containing functional groups, nanoscale capillary channels, surface defects, and charge contribute to water retention and water transport properties and to organ-specific growth responses at the soil–plant interface [8,10].

After application, the effective exposure of plants to GO depends strongly on dispersibility and aggregation behavior in the receiving matrix. In aqueous media and soil porewater, pH and ionic composition regulate surface charge and electrostatic screening, and divalent cations, such as Ca²⁺ and Mg²⁺, can promote aggregation and retention via charge neutralization and cation bridging. In contrast, natural organic matter, including humic substances, may enhance dispersibility through steric and electrosteric stabilization and can increase transport in porous media, thereby modifying the probability of GO contacting root surfaces. In soils, additional interactions with mineral surfaces, organic matter, and microbial products further control deposition, persistence, and biotransformation. GO can undergo time-dependent changes in dispersion state and surface reactivity, including corona formation and redox or aging processes, which may shift the balance between direct surface interactions, such as physical contact and ion adsorption at the biointerface, and indirect effects mediated by changes in water retention, nutrient availability, and rhizosphere microenvironments [10,14]. For this reason, GO plant studies should report not only nominal application dose but also key matrix descriptors, such as pH, ionic strength, dominant cations, and organic matter content, together with basic dispersion metrics, to improve cross-study comparability and mechanistic interpretation.

3. Effects of GO on Plant Growth and Development

3.1. Seed Germination

To improve mechanistic clarity, we distinguish two modes of action throughout this review. Direct GO plant interactions refer to physicochemical contacts at the seed, root, or leaf interface, including surface deposition, adsorption of ions, membrane perturbation, and intracellular entry that can directly trigger oxidative stress or disrupt photosynthetic machinery under controlled exposure. Indirect effects refer to microenvironment-mediated pathways, where GO changes water retention, ion availability, nutrient transport, or rhizosphere processes, and the observed shifts in hormone-related genes or antioxidant enzymes are secondary responses to improved or perturbed resource status. In many studies, these two modes may co-occur, and the dominant pathway depends on exposure route, dose, and the surrounding matrix.

GO itself does not provide nutrients, but it can markedly influence the germination process through its particular capacity for water handling. In soil systems, application of 50 mg/L GO significantly increased the germination percentage of Spinacia oleracea L. and Allium schoenoprasum L. and shortened the time to emergence, whereas treatment with 200 mg/L shifted the effect from promotion to inhibition, as reflected by reduced germination and delayed emergence [8]. These findings indicate a typical concentration-dependent response of seed germination to GO. Previous studies have suggested that high doses of GO may disturb the dynamic balance of water between the exterior and interior of the seed coat because of its strong hydrophilicity, and that its inhibitory effects on soil microbial communities may weaken the beneficial microbial functions required for successful germination. It is noteworthy that the germination-promoting effect of GO often depends on sufficient soil fertility. In Amorpha fruticosa L. and Festuca arundinacea Schreb., low doses of GO consistently increased germination and early seedling growth only under relatively adequate nutrient supply, whereas the effect was markedly reduced or not significant under nutrient-poor or nutrient-limited conditions [9,10]. These observations suggest that GO acts more as a regulator of water availability and microenvironmental conditions rather than as a nutrient source that can replace fertilizers. In this context, nano-enabled seed priming has emerged as a promising strategy to precondition crops for enhanced stress resilience without altering their genetic background, and carbon-based nanomaterials, particularly GO-based priming, are now attracting increasing attention as model systems in this field [14,15].

More recently, a multi-omics integration study on Arachis hypogaea L. provided a more systematic view of GO-based seed priming. Seed priming with 400 mg/L GO promoted germination by modulating internal carbon and nitrogen metabolism and secondary metabolite biosynthesis. It also systemically activated several salt-tolerance mechanisms at the seedling stage [16]. Importantly, GO itself was not detected in leaves, and its systemic effects were therefore attributed to signaling processes initiated during the priming phase rather than to direct accumulation in aerial tissues. These findings, alongside classical research on osmopriming, indicate that GO-based nano priming may reprogram central carbon and nitrogen metabolism, with potential implications for epigenetic markers, thereby establishing a form of stress memory that can be activated during later developmental stages [17]. This work reveals a new mode of action in which GO functions as a priming agent that induces systemic resistance in plants. It extends the current understanding that GO influences germination not only through physical regulation of water availability but also through possible epigenetic or metabolic reprogramming that establishes a form of stress memory and enhances plant adaptation under adverse conditions. Recent work in Arabidopsis thaliana (L.) Heynh. lines carrying the bar marker gene further showed that low-dose GO priming between 0.75 and 1.5 mg C/L enhanced chlorophyll content and photosynthetic efficiency in wild-type plants but aggravated lipid metabolism disorders and downregulation of jasmonic-acid signaling in a transgenic line with a naturally altered alpha linolenic acid pathway, and that GO exposure induced stronger DNA methylation dynamics in alpha linolenic acid metabolism genes in the transgenic line that were partly transmitted to the F1 generation, which highlights that genotype-specific responses and preexisting genetic variation can strongly shape the outcomes of GO based seed priming [18].

3.2. Root System Architecture and Leaf Growth

The regulatory effects of GO on plant growth and development show clear concentration dependence and species specificity and involve complex interactions among hormone networks, antioxidant systems, and transcriptional reprogramming. In herbaceous species, GO coordinates the reconstruction of root system architecture mainly by modulating signaling of indole-3-acetic acid (IAA) and abscisic acid (ABA). In Nicotiana tabacum L., treatment with 20 mg/L GO suppresses primary root elongation while promoting the formation of adventitious roots [19]. At the same time, it activates the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and upregulates auxin-responsive genes, including IAA3, IAA4, and auxin response factor 2 (ARF2), which together alleviate oxidative damage and optimize root system architecture. Studies in Solanum lycopersicum L. further highlight the importance of endogenous hormone status [20,21]. The same concentration of GO promotes primary-root elongation in wild-type plants but inhibits growth in ABA overexpressing lines, indicating that the response to GO depends on the crosstalk between ABA and IAA signaling. In Brassica napus, GO reduces IAA content and increases ARF2 expression, whereas in Malus domestica (Suckow) Borkh., it promotes adventitious root formation by activating the antioxidant system and regulating genes, such as ARRO1 and TTG1, but it restricts root elongation by downregulating auxin transport carriers such as PIN7, which underlines the species-specific nature of the regulatory networks involved [22,23].

In grasses and woody species, the growth-promoting effects of GO are also mediated through multiple pathways. In Triticum aestivum L., low concentrations of GO at 100 mg/L induce mild oxidative stress that activates the antioxidant system and increases root length and biomass by 20 to 68 percent, whereas a high concentration of 1000 mg/L causes severe lipid peroxidation and disruption of ion homeostasis [24]. In Oryza sativa L., GO interferes with IAA metabolism and thereby induces the reconstruction of root systems [25]. In a sensitive cultivar, IAA content increases by about 35 percent, but root length is suppressed, while in a tolerant cultivar, the number of lateral roots increases by about 19 percent, which shows that the response to GO is closely linked to cultivar-specific hormone networks. Taken together, these cereal studies indicate that GO-mediated modulation of oxidative status and auxin signaling is a common mechanism underlying changes in root system architecture.

It is noteworthy that the regulation of leaf growth by GO is often achieved indirectly through coordinated changes from roots to leaves. In Aloe vera (L.) Burm.f., treatment with 50 mg/L GO mainly relies on improvements in water and nutrient uptake that result from optimized root structure [26]. These changes enhance leaf photosynthetic performance and promote the accumulation of proteins, amino acids, and other nutrients, while no adverse effects on the levels of key medicinal components have been detected, which indicates a relatively clear tissue-targeting pattern. Overall, GO reshapes root system architecture through combined biointerface effects and microenvironment-mediated changes in water and nutrient status, and the observed shifts in hormone signaling and antioxidant defenses should often be interpreted as downstream responses unless direct molecular targets are demonstrated. Its net biological effects are jointly determined by crop species, genetic background, exposure route, and dose.

3.3. Reproductive Growth and Yield Quality

GO and graphene-based nanomaterials exert multi-level and multi-target effects on reproductive growth and yield quality. In general, these carbon-based nanomaterials can alter floral organ differentiation and fruit expansion, and they are often associated with changes in carbon and nitrogen metabolism and stress defense systems. In many cases, these molecular and biochemical shifts may reflect indirect improvements in water and nutrient status rather than direct receptor-level interactions. In A. thaliana, treatment with low-to-medium concentrations of GO in the range of 0.1 to 10 μg/L does not cause obvious cytotoxicity. During the vegetative phase, GO has no significant effect on leaf area, fresh weight, root length, or leaf number, whereas during the reproductive phase, it markedly increases the number of floral buds. At higher treatment levels of 1 and 10 mg/L, the number of floral buds reaches 11.67 and 11.33, respectively, which is clearly higher than in the control, indicating that GO can promote the flowering process without compromising vegetative growth [27].

In fruit crops, GO application increases the fruit circumference of Citrullus lanatus (Thunb.) Matsum. & Nakai by about 5 cm compared with the control and raises the Brix value by 1.57, which suggests that GO favors both fruit expansion and the accumulation of soluble sugars and therefore has potential as a functional nanoregulator for improving yield and quality in fruit and vegetable crops [27]. Reproductive endpoints can represent a high sensitivity window for GO, particularly in pollen-related processes. Therefore, applications near flowering should avoid direct contact with floral tissues and should rely on conservative dose validation with reproductive performance endpoints [28].

Beyond GO, other graphene-based nanomaterials also show promise in the regulation of plant growth and quality. For example, appropriate doses of Graphene (GN) promote biomass accumulation in medicinal plants and increase the contents of artemisinin and other secondary metabolites in Artemisia annua L. [29]. Graphene quantum dots (GQDs) enhance biomass and chlorophyll accumulation in N. tabacum and, at the same time, improve antiviral capacity [30]. In S. lycopersicum, foliar spraying or root drenching with GN strengthens antioxidant defense and increases the accumulation of photosynthetic pigments [31]. These findings on non-oxidized graphene systems support the broader view that carbon-based nanomaterials have considerable potential for increasing yield, improving quality, and stabilizing production, and they provide additional evidence that can inform the extension of GO-based applications in medicinal and high-value crops.

3.4. GO-Mediated Responses in Woody Plants

3.4.1. Establishment and Stress Resilience in Woody Crops and Shrubs

GO and related graphene-based materials show pronounced dose dependence and species specificity in woody plants. Compared with annual crops, woody systems are characterized by longer life spans, repeated exposure scenarios, and management goals that prioritize establishment and long-term resilience. To clarify the evidence base, we separated studies in woody crops and agroforestry shrubs from those in forest trees, and we summarized benefits for establishment and stress tolerance together with reproductive stage sensitivities.

At the establishment stage, moderate GO can promote root branching and adventitious rooting in woody crops. In M. domestica, GO increases the number and total length of adventitious roots, accompanied by elevated SOD, POD, and CAT activities and by upregulation of ARRO1 and TTG1, while downregulating the auxin transporter PIN7, resulting in enhanced branching with limited primary root elongation. In woody berry crops, GO- or graphene-based treatments can also promote fine root development. In R. idaeus, graphene treatment almost doubles both root tip number and specific root area and is accompanied by higher antioxidant enzyme activities [32]. Collectively, these observations suggest that cell-wall-associated interactions and hormone–redox regulation can converge to accelerate root establishment in woody plants.

Under abiotic stress, GO-mediated benefits in woody systems are often indirect and rhizosphere-driven. In P. ostii, soil drenching with 80 to 100 mg/L GO as a water retention agent effectively suppresses soil water loss and increases relative water content in the root zone [33]. Under drought stress, this treatment helps to maintain leaf photosynthetic efficiency and cellular ultrastructure, while spectroscopic and anatomical data indicate that GO remains largely in the soil and does not accumulate in plant tissues, suggesting an indirect effect through physical improvement of the rhizosphere. In the grapevine V. vinifera ‘Sultana’, soil application of nano-GO combined with foliar sprays of nano-Fe or nano-Se under 100 mM NaCl significantly increases shoot dry weight and chlorophyll a and b contents. It also enhances the activities of SOD and CAT and improves the uptake of nutrients such as K⁺ and Ca²⁺, thereby alleviating salt-induced inhibition of growth and photosynthetic machinery [34]. Studies on woody legumes, such as A. fruticosa, further show that low-dose GO applied as seed dressing or soil amendment can increase germination rate and seedling biomass when nutrient supply is adequate, whereas the effect becomes weak or negligible in nutrient-poor soils. This supports the view that GO acts mainly as a modulator of the microenvironment rather than as a direct nutrient source.

However, GO may pose specific risks during reproductive stages in woody plants. In hazel Corylus avellana L., 100 mg/L GO reduced pollen germination by about 83 to 84 percent and markedly inhibited pollen tube elongation [28]. Mechanistic analyses suggest that GO lowers the pH of the germination medium and strongly adsorbs Ca²⁺, while disturbing the tip-focused reactive oxygen species (ROS) gradient in pollen tubes, thereby disrupting ion balance and redox homeostasis required for tip growth. Compared with few-layer graphene and reduced GO, GO with abundant oxygen-containing functional groups showed stronger inhibitory effects on pollen performance.

Taken together, GO and related graphene materials can enhance root establishment and mitigate drought and salinity stress in woody plants, whereas reproductive stage endpoints may represent a high sensitivity window. Therefore, practical applications should prioritize pre-flowering scenarios, such as seed dressing, soil amendment, or early vegetative treatments, and avoid direct flower contact; if flowering stage use is unavoidable, conservative dose–response testing should target pH, Ca²⁺ availability, and ROS-related reproductive endpoints (Figure 1).

3.4.2. Long-Term Evidence on Nursery and Propagation of Forest Trees

Evidence in forest trees remains limited but is expanding from seedling assays to tissue-culture models. Because forest trees are long-lived, evaluation should extend beyond short-term growth to include GO persistence in nursery substrates, potential accumulation in woody tissues, and multi-season carry-over effects, together with rhizosphere-mediated biotransformation. Soil persistence is a key uncertainty for forestry-oriented uses because repeated inputs and substrate aging may change GO retention and bioavailability over time. Therefore, forestry evaluations should include soil fate endpoints, including aging and half-life assays, vertical transport and leaching potential, and multi-season carry-over effects on seedlings and rhizosphere function. Because forest productivity and seedling establishment often depend on mycorrhizal symbioses, forestry-oriented assessments should also track mycorrhizal colonization and ectomycorrhizal community shifts, particularly in restoration substrates.

In the conifer P. tabuliformis, exposure to about 25 mg/L graphene-based nanomaterials led to an increase in the number of root tips by approximately 239 percent [35]. Transcriptome analysis showed activation of pathways related to abiotic-stress responses and the cell cycle, together with partial suppression of biotic-defense pathways, indicating that carbon nanomaterials can promote root system establishment in conifers by modulating cell division and stress signaling [35]. In Populus nigra L. callus cultures, a three-week exposure to GO at 25 to 101 mg/L elicited sex-specific adaptive responses, indicating that genetic background can shape GO sensitivity and should be considered when screening planting material [36]. In Betula pubescens Ehrh. microclones, GO at 0.75 to 15 μg/L displayed a hormetic pattern, with stimulation at 1.5 to 3 μg/L but inhibition at 15 μg/L, including an approximately 20 percent reduction in shoot height at the higher dose [37]. Together, these studies support the need for forest-specific dose windows and propagation-relevant endpoints when translating graphene and graphene oxide applications from crops to forestry.

4. Roles of GO-Based Nanomaterials in Plant Stress Responses

4.1. Abiotic Stress

4.1.1. Salt Stress

GO enhances plant salt tolerance through several complementary mechanisms and can be applied in different ways [38]. Under soil application conditions, low concentrations of GN supplied to Medicago sativa L. do not enter plant tissues but instead improve the rhizosphere environment. This treatment helps maintain ion homeostasis, activates antioxidant enzymes, and protects the photosynthetic apparatus under 120 mM salt stress, which effectively alleviates salt-induced damage [39,40]. Similar effects have been observed in Silybum marianum (L.) Gaertn., where soil application of GO induces the accumulation of osmolytes such as proline, enhances antioxidant capacity, and improves the quantum efficiency of photosystem II (PSII), thereby mitigating salt stress-induced growth inhibition [41].

Foliar application mainly acts through more direct regulation of photosynthesis and antioxidant function. In Fragaria × ananassa (Duchesne ex Weston) Duchesne ex Rozier, spraying with low concentrations of GO at 5 to 10 mg/L markedly increases tolerance to saline alkaline conditions. Chlorophyll fluorescence analysis based on OJIP transients indicates that GO improves PSII performance, stabilizes electron transport, and increases light energy conversion efficiency, which ultimately enhances gas exchange parameters and biomass accumulation [42].

Building on these findings, functionalized GO provides a materials strategy to improve salt tolerance and grain nutritional quality at the same time. Methionine- and lysine-functionalized GO significantly enhance the nutritional quality of grains in Pennisetum glaucum (L.) R.Br. under salt stress [43]. This improvement is closely associated with the stimulation of key enzymes involved in nitrogen assimilation, the reconfiguration of the tricarboxylic acid (TCA) cycle, and the resulting accumulation of amino acids and organic acids, together with salt-related epigenetic reprogramming and redistribution of phenylpropanoid metabolism.

When combined with transcriptomic evidence, these results suggest that graphene-based materials can also modulate salt tolerance at the molecular level by regulating key transcription factors such as APETALA2 (AP2) and ethylene response factor (ERF), which in turn control gene expression in pathways related to photosystem antenna proteins and glutathione metabolism and thus provide additional mechanistic support for their roles in salt-stress regulation [44]. Consistent with these findings, a GO glycine betaine conjugate at 50 to 100 mg/L more effectively reversed growth inhibition of Ocimum basilicum L. under 50 to 100 mM NaCl than GO or glycine betaine alone by increasing photosynthetic pigment content and chlorophyll fluorescence parameters, stabilizing membranes, enhancing activities of antioxidant enzymes, such as ascorbate peroxidase, superoxide dismutase and guaiacol peroxidase and promoting the accumulation of proline and phenolic compounds while optimizing essential oil composition, and pure GO at 100 mg/L showed clear toxicity whereas GO glycine betaine at the same concentration did not, which indicates that functionalization with compatible solutes can mitigate GO phytotoxicity and at the same time strengthen its protective effects under salinity [45]. Taken together, these studies indicate that GO and its functional derivatives mitigate salinity damage not only by buffering ion imbalance and oxidative stress, but also by reshaping salt-responsive networks at the transcriptomic, metabolomic, and possibly epigenetic levels.

4.1.2. Other Abiotic Stresses

Under heavy metal stress, GO can either alleviate toxicity or amplify risk, depending on the exposure context. In aqueous systems, GO exhibits a maximum adsorption capacity of 23.4 mg/g for Cu²⁺, which effectively immobilizes Cu ions in solution and mitigates growth inhibition and oxidative damage in Lemna minor L. [46,47]. Consistently, a carboxylated GO chitosan composite bead showed a Cu²⁺ adsorption capacity of about 78 mg/g and reduced Cu concentration in soil leachate to below 1.0 mg/L, which meets drinking water standards, and when applied to contaminated soil it lowered Cu accumulation in wheat roots by about 60 percent without inhibiting seedling growth, indicating that functionalized GO materials can immobilize heavy metals in soil and reduce metal uptake by crops [48]. In a foliar application study, low concentration GO at 30 mg/L chemically fixed Cd²⁺ through its surface oxygen-containing functional groups and formed a partial physical barrier and systemic regulatory effect in Lactuca sativa L., which significantly reduced Cd accumulation and improved product quality [49].

In contrast, soil application of a high dose of GO at 3 g/kg altered Cd speciation by converting less available inorganic bound forms into more bioavailable exchangeable forms and markedly increased Cd uptake by O. sativa, indicating that GO may pose a potential risk of activating heavy metals in soil environments [50]. Immobilization is more likely at low to moderate doses when GO is functionalized or used as a composite sorbent that reduces free ion activity. Mobilization becomes more likely under high soil doses when GO shifts metal speciation toward exchangeable pools, and this direction is strongly conditioned by soil pH, dissolved organic matter, and application route. Therefore, the expected outcome should be interpreted jointly by dose and route, GO surface chemistry, and soil matrix properties. However, green synthesized reduced GO at 30 mg/L mitigated the inhibitory effects of 700 mg/L Pb on wheat by increasing root length and plant height by about 17.5 and 6.8 percent and fresh weight by about 14.1 percent, improving chlorophyll content, enhancing antioxidant enzyme activities and halving Pb accumulation in roots, which supports a three level mitigation mechanism that combines Pb adsorption by the porous reduced GO structure, activation of antioxidant defense and upregulation of stress tolerance genes [51].

For non-metal stresses, the role of GO in drought mitigation mainly depends on physical improvement of the rhizosphere environment [52]. Soil drenching with low concentrations of GO at 80 to 100 mg/L, supported by its strong hydrophilicity, suppresses soil water evaporation and increases relative soil water content, thereby providing more favorable water conditions in the rhizosphere. These changes enhance antioxidant capacity, improve photosynthetic efficiency, and help maintain cellular structural integrity in drought-stressed crops such as Setaria italica (L.) P.Beauv. [53]. In addition, sulfonated GO at 50 to 500 mg/L alleviated inhibition of photosystem II in T. aestivum leaves exposed to 140 mM NO₃⁻ and 5 mM NH₄⁺ by increasing the maximum quantum efficiency Fv/Fm and performance index and by upregulating photosystem genes such as psaA and psbA, while activation of the ascorbate glutathione cycle together with enhanced superoxide dismutase and peroxidase activities reduced H₂O₂ accumulation and lipid peroxidation in chloroplasts with low-to-medium doses relying mainly on ascorbate glutathione metabolism and higher doses depending more on peroxidase mediated detoxification, which shows that functionalized GO can also support chloroplast function and redox homeostasis under nitrogen stress [54]. Under drought conditions in soybean, GO treatment increased relative water content in stems and leaves by about 127 and 128 percent and enhanced root system traits such as total length, surface area, average diameter and volume by 33, 38, 34, and 35 percent, while activation of antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase together with reduced malondialdehyde and H₂O₂ accumulation and upregulation of drought responsive genes such as GmP5CS, GmGOLS, GmDREB1, and GmNCED1 and increased levels of jasmonic acid, salicylic acid, and abscisic acid demonstrated multilayer regulation of soybean drought tolerance by GO [55].

When GO coexists with environmental pollutants, its overall impact can become more complex and may lead to synergistic toxicity. In cultures of Microcystis aeruginosa (Kützing 1833) Kützing 1849, low concentrations of GO below 10 mg/L show weak intrinsic toxicity but strongly enhance Cd toxicity by adsorbing and concentrating Cd²⁺ and delivering it to the algal cell surface and interior [56]. In O. sativa, although a high concentration of GO at 400 mg/L can downregulate the expression of root Cd transporter genes and thereby reduce Cd accumulation in plant tissues, the severe toxicity induced by GO itself offsets or even masks this detoxification effect and ultimately results in more pronounced growth inhibition [57]. Overall, the net effects of GO on plant responses to abiotic stress depend on the environmental matrix, application dose, and exposure route. Future work should define safe concentration ranges and operational boundaries for GO in different combinations of environmental conditions and biological systems, and should include long-term and combined stress scenarios in ecological risk assessment [58]. Such assessments should report soil aging and half-life behavior, vertical transport and leaching potential, and tissue residues across roots and shoots, including edible organs when relevant. Bioaccumulation and trophic transfer should be evaluated with soil invertebrate and microbial community endpoints under repeated exposure designs that match realistic use patterns. A standardized test battery can further include earthworm survival and reproduction, microbial community sequencing and functional assays, and multi-season field or nursery trials under repeated application scenarios.

4.2. Interactions with Soil Microorganisms

GO- and graphene-based nanomaterials influence plant–microbe interactions mainly through two types of processes. The first is the modulation of rhizosphere microbial community structure and function, which affects nutrient cycling and plant growth. The second is the direct multi-target inhibition of plant pathogenic microorganisms. Compared with the earlier view that these materials act as simple broad-spectrum antimicrobial agents, current evidence supports their role as regulators of microecological processes. In L. sativa, Das showed that graphene nanoadditives significantly increase the abundance of plant growth-promoting bacteria, such as Microvirga, in the rhizosphere, decrease the gene abundance of ammonia-oxidizing bacteria (amoA), and enhance the activities of key enzymes involved in the carbon, phosphorus, and sulfur cycles [59]. These changes improve nitrogen-use efficiency and promote biomass accumulation. In the rhizosphere of A. annua, graphene treatment similarly enriches Acidobacteria, Actinobacteria, and multiple taxa associated with organic matter decomposition.

Under environmentally relevant concentrations from 1 ng/kg to 1 mg/kg, Forstner further demonstrated that GO does not markedly alter soil microbial alpha diversity but does reshape the composition of bacterial and fungal communities [60]. Its effects are comparable to those of bulk graphite and do not follow a simple dose–response pattern, which suggests that GO mainly drives community reorganization and functional redistribution through physicochemical processes such as surface adsorption and sheet aggregation rather than by uniformly suppressing microbial activity. Beyond community level shifts, hydroponic experiments in O. sativa showed that GO treatment reduced the abundance of antibiotic resistance genes in roots and shoots by about 71 and 86 percent and decreased the complexity of the endophytic bacterial co-occurrence network, while suppression of biofilm formation and mobile genetic elements further limited horizontal transfer of resistance determinants, which indicates that appropriate GO doses may help to constrain the spread of antibiotic resistance genes in agroecosystems although the balance between this benefit and potential phytotoxicity still requires careful evaluation [61]. Taken together, these studies indicate that GO and related carbon-based materials promote rhizosphere microecological stability and nutrient utilization primarily by optimizing microbial community structure and function and thus provide a mechanistic basis for their potential use in agricultural microecology management and ecological risk assessment. Accordingly, GO-induced reorganization of rhizosphere microbial communities should be regarded as part of its indirect biostimulant action rather than as a simple antimicrobial toxicity, which has important implications for defining its ecological risk profile and agronomic application window.

For pathogen control and materials design, GO and its functional derivatives exhibit characteristic multi-target antimicrobial activity. Studies on Fusarium graminearum Schwabe, 1839. show that GO inhibits both growth and virulence in a concentration-dependent manner. Multi-omics analyses indicate that GO interferes with the synthesis of cell wall-associated proteins, reprograms carbon and nitrogen metabolism, and suppresses the production of nucleic acid precursors, thereby weakening fungal nutrient metabolism and stress defense at several levels [62].

For bacterial pathogens, GO at 250 mg/L killed about 95 percent of Xanthomonas oryzae (ex Ishiyama 1922) Swings et al. 1990 pv. oryzae cells that cause O. sativa, bacterial leaf blight, whereas a conventional thiodiazole copper formulation achieved only about 13.3 percent mortality under comparable conditions, which demonstrates that GO can act as a highly effective nano bactericide for O. sativa disease management [63]. Building on this, Garwal used agricultural waste as a precursor to synthesize potassium-doped GO (K-GO) in a one-step process and obtained a material with broad-spectrum, concentration-dependent inhibitory effects on diverse bacterial and fungal strains. The antibacterial mechanism of K-GO combines enhanced electrostatic adsorption to negatively charged cell surfaces with aggravated membrane damage and the induction of ROS accumulation, while avoiding the environmental risks associated with conventional preparation methods that rely on strong acids and strong oxidants [64]. In a complementary approach, a gallic acid-loaded GO nanocomposite achieved a loading efficiency of about 76 percent and inhibited methicillin resistant Staphylococcus aureus Rosenbach 1884 at 300 mg/L, whereas free gallic acid required 500 mg/L for comparable inhibition, which indicates a clear synergistic enhancement of antibacterial activity by the GO carrier and suggests that GO-based nanoformulations can improve the delivery and efficacy of natural phenolic antimicrobials relevant to plant disease control. In addition to direct antimicrobial activity, GO can also enhance crop resistance to insect herbivores by modulating endogenous immune signaling. In Sorghum bicolor (L.) Moench, foliar application of 10 mg/L GO reduced aphid populations and improved plant growth by combining nanozyme-like scavenging of aphid-induced ROS bursts with the suppression of miR319 and activation of the transcription factor SbTCP7 and the jasmonic acid biosynthesis gene SbLOX3, which, together, stimulated jasmonic acid-dependent defense responses [65]. These functional carbon-based nanomaterials centered on GO suggest that it is possible to balance antimicrobial efficacy with environmental compatibility and that such materials may provide new options for crop disease management and maintenance of soil health (Figure 2 and

Table 1. Applications of graphene oxide in plant growth and stress tolerance enhancement.

Plant	Treatment Material	GO Concentration	Treatment Time (Days)	Observed Effects	Ref.
Spinacia oleracea; Allium schoenoprasum	seeds	50 mg/L	40; 72	Significantly promoted germination at a concentration of 50 mg/L, which represents the lowest observed effective level for this beneficial effect.	[8]
Amorpha fruticosa	seeds	50–200 mg/L	10	Promoted germination and seedling growth across a broad concentration range (50–200 mg/L), with an estimated NOAEL < 50 mg/L.	[16]
Festuca arundinacea	seeds	0.2 mg/L	10~30	Promoted seed germination and enhanced biomass at a very low concentration (0.2 mg/L), identified as the lowest observed effective level.	[17]
Arachis hypogaea	seeds	400 mg/L	6	Improved osmotic regulation and metabolism, resulting in a significant yield increase at 400 mg/L, which is the effective concentration threshold for benefit.	[20]
Vicia faba	seeds	400 and 800 mg/L	Until the seeds germinated	Improved plant growth via enhanced water absorption and antioxidant defense at concentrations of 400 and 800 mg/L, indicating a beneficial effect LOEL of 400 mg/L.	[66]
Nicotiana tabacum	seedlings	20 mg/L	35	Significantly increased adventitious root length and alleviated oxidative stress at 20 mg/L, with this concentration serving as the NOAEL for the observed beneficial effects.	[23]
Solanum lycopersicum	seedlings	20 mg/L	15	Significantly increased seminal root length and alleviated oxidative stress at 20 mg/L, defined as the effective beneficial concentration.	[25]
Brassica napus L.	seedlings	50 mg/L	15	Induced changes in root growth and phytohormone (ABA, IAA) levels at 50 mg/L, representing the threshold concentration for physiological effects.	[26]
Malus domestica	seedlings	0.1 mg/L	40	Exhibited a biphasic effect: positively influenced root formation but negatively affected root elongation at the same concentration (0.1 mg/L), indicating a very low LOAEL for adverse effects on root growth.	[27]
Triticum aestivum	seedlings	100 mg/L	14	Promoted root growth at 100 mg/L, establishing this as the lowest observed beneficial concentration.	[28]
Oryza sativa	seedlings	5–50 mg/L	5	Significantly affected root development within the 5–50 mg/L range, with the lowest concentration (5 mg/L) representing the threshold for observable effects.	[29]
Pinus tabuliformis Carrière	seedlings	25 mg/L	180	Significantly promoted root growth at 25 mg/L, indicating this as the effective beneficial dose.	[36]
Rubus idaeus L.	seedlings	2 mg/L	30	Increased seedling height by 1.46-fold and approximately doubled root length at 2 mg/L, which is the effective concentration for growth promotion.	[37]
Vitis vinifera L.	seedlings	50,000 mg/kg soil	75	Enhanced salinity tolerance mechanisms at a high soil dose of 50,000 mg/kg, which was the effective level for benefit under stress conditions.	[39]
Arabidopsis thaliana	seedlings	2 and 4 mg/L	14	Displayed a genotype-dependent response: promoting growth in WT plants at 2 mg/L (Beneficial LOAEL) but inhibiting growth in GM plants at the same concentration (Adverse LOAEL), amplifying metabolic mutations.	[22]
Arabidopsis thaliana	seedlings	1 mg/L	30	Significantly increased the number of flower buds at 1 mg/L, indicating a very low effective concentration for this reproductive effect.	[31]
Lemna minor	seedlings	5 mg/L	4	Alleviated copper stress and promoted growth at 5 mg/L, defining the concentration threshold for stress mitigation.	[50]
Setaria italica	seedlings	80 mg/L	15	Alleviated drought-induced growth inhibition and increased ear weight (54.73%) at 80 mg/L, the effective dose for drought resilience.	[10]
Microcystis aeruginosa	seedlings	400 mg/L	3~14	While reducing Cd accumulation, it aggravated growth inhibition at 400 mg/L, which is therefore the LOAEL for the adverse effect under cadmium stress.	[60]
Betula pubescens	microclones	0.00075–0.015 mg/L	46	Exhibited a clear biphasic (hormetic) response: shoot and leaf growth were promoted at low concentrations (0.0015–0.003 mg/L, with a NOAEL ~0.00075 mg/L) but inhibited at the highest concentration tested (0.015 mg/L, LOAEL for adverse effects). Inhibited shoot growth	[41]
Solanum lycopersicum	seedlings and mature plants	50 mg/L and 100 mg/L	30	Increased root auxin content, fruit yield, and accelerated ripening at both 50 and 100 mg/L, with no adverse effects observed, suggesting a NOAEL of at least 100 mg/L for these beneficial traits.	[24]
Aloe vera	seedlings and leaves	50 mg/L	120	Improved photosynthesis, yield, and nutrient content at 50 mg/L, the effective concentration for these positive outcomes.	[30]
Citrullus lanatus	seedlings	10 mg/L	30	Enhanced fruit expansion rate and sugar content at 10 mg/L, representing the effective dose.	[31]
Paeonia ostii T.Hong & J.X.Zhang	mature plants	0.1 mg/L	18	Significantly improved soil water retention and plant drought tolerance at 0.1 mg/L, the threshold concentration for this effect.	[38]
Medicago sativa	seedlings	5000 mg/kg soil	46	Activated antioxidant defense and photosynthesis-related genes at a soil dose of 5000 mg/kg, indicating the effective level.	[43]
Silybum marianum	seedlings	N/A	72	Improved photosynthetic performance and alleviated salt stress damage; however, a clear concentration threshold cannot be defined from the available data.	[45]
Fragaria × ananassa	leaves	5–10 mg/L	160	Increased pigment content, biomass, and early yield within the 5–10 mg/L range, with 5 mg/L as the lowest effective concentration.	[46]
Pennisetum glaucum	leaves	20 mg/L	30 and 60	Alleviated oxidative damage, improved nutritional quality, and increased yield at 20 mg/L, the defined beneficial concentration.	[47]
Lactuca sativa	leaves	30 mg/L	28	Significantly reduced Cd accumulation and promoted growth at 30 mg/L, the effective concentration for these combined benefits.	[67]
Sorghum bicolor	leaves	10 mg/L	16	Significantly reduced aphid population and improved plant fresh weight and height at 10 mg/L, establishing this as the effective dose for pest resistance and growth enhancement.	[68]
Populus nigra	callus	25–100 mg/L	21	Showcased sex-specific responses: females were tolerant, showing increased biomass even at 100 mg/L (NOAEL), while males were sensitive, exhibiting growth inhibition at the lowest concentration tested of 25 mg/L (LOAEL).	[40]

).

5. Toxicity Mechanisms of GO in Plants

5.1. Dose-Dependent Responses and Early-Stage Sensitivity

GO and related graphene-based materials exhibit dose-dependent and structure-specific phytotoxicity. At low concentrations, GO can induce a hormetic response in certain species and developmental stages, characterized by stimulatory effects at low doses that shift to inhibition at higher doses. For example, in Vicia faba L., moderate GO application improves seed water uptake and moderately induces antioxidant enzyme activities, which promote germination and early seedling growth [69]. In O. sativa and T. aestivum, treatment with 100 to 200 mg/L GO does not markedly affect germination percentage, but at the seedling stage, low concentrations can slightly stimulate root elongation, whereas higher concentrations significantly inhibit growth [70]. A comparative germination assay with L. sativa, Raphanus sativus L., Lolium perenne L., M. sativa, and Cucumis sativus L. further showed that GO at concentrations up to 1600 mg/L exerted the strongest toxicity on L. sativa, where shoot and root length decreased by about 87 and 86 percent, respectively. In contrast, germination of the other species was less affected, although seedling growth of M. sativa and R. sativus still declined, which highlights pronounced species-specific differences in sensitivity to high GO doses during germination and early growth [71].

5.2. Root-Centered Toxicity and Cellular Stress Mechanisms

The root system is often a primary target of GO toxicity under root exposure scenarios. On the one hand, GO readily forms a coating layer on the root surface, which leads to root tip shrinkage, epidermal peeling, and cell necrosis, and suppresses the formation of lateral roots and root hairs. These changes directly weaken water and nutrient uptake. At the same time, GO downregulates the expression of nitrate transporter genes, such as NRT2.1 and NRT2.3 in T. aestivum roots, reduces net NO₃⁻ uptake, and decreases nitrogen accumulation in the plant [68]. GO also acidifies the nutrient solution, enhances the mobilization and excessive accumulation of iron and other elements, and thereby aggravates oxidative damage [66]. On the other hand, plants can partially reduce GO to less toxic, reduced graphene oxide (rGO) through redox-active root exudates, suggesting a potential intrinsic detoxification route. Stable isotope tracing with ¹³C-labeled GO in hydroponically grown T. aestivum seedlings quantified pronounced accumulation of GO in roots at about 112 μg/g fresh weight after 15 days of exposure to 1000 mg/L, with only limited translocation to shoots. This root retention coincided with disruption of tissue organization, induction of oxidative stress, and inhibition of germination and early growth, which provides direct evidence that persistent root-associated GO may pose ecological risks in cereal crops [72].

At the physiological level, oxidative stress and ion homeostasis disruption are central to GO-induced phytotoxicity. High concentrations of GO cause extensive generation of ROS. This initially activates antioxidant enzymes such as SOD, POD, and CAT, but prolonged exposure still leads to marked accumulation of the lipid peroxidation product malondialdehyde (MDA). In parallel, ROS attack and GO-induced inhibition of membrane proteins, such as plasma membrane H⁺-ATPase, jointly trigger membrane depolarization and ion transport disorders, disrupt intracellular pH homeostasis and calcium signaling, and, ultimately, result in metabolic dysfunction at the cellular level. In Fagopyrum esculentum Moench, integrated physiological and transcriptomic analysis showed that high GO concentrations reduced seedling growth and induced ROS accumulation, altered catalase and peroxidase activities and suppressed the expression of genes involved in hormone biosynthesis and signaling, and identified 97 small secreted peptide genes together with interaction networks involving 111 transcription factors and 43 receptor like kinases that respond to GO, which outlines a small secreted-peptide-centered signaling framework for buckwheat adaptation to GO stress [67]. In many cases, changes in hormone pathways and antioxidant systems likely represent downstream stress responses that follow altered water, nutrient, or ion status, rather than direct molecular interactions between GO and specific signaling receptors.

5.3. Material Properties, Translocation, and Implications for Food Safety

The physicochemical properties of GO strongly influence its toxicity pattern and target sites. Single-layer GO (sGO), which has a smaller lateral size and higher specific surface area, more readily induces oxidative damage and repression of stress-related genes in roots than multi-layer GO (mGO) [73]. In contrast, rGO shows lower direct contact toxicity to roots under hydroponic conditions, but its increased hydrophobicity greatly enhances translocation from roots to shoots. Studies using ¹³C-stable-isotope labeling have demonstrated that rGO is more readily transported between organs in Pisum sativum L., accumulates in leaves, and directly inhibits photosynthesis by damaging the oxygen-evolving complex (OEC) on the donor side of PSII [74]. The OEC catalyzes water splitting and supplies electrons to PSII. Thus, its impairment blocks electron flow and lowers PSII efficiency. These findings indicate that the reduced form of graphene-based materials may alleviate local oxidative injury in roots while increasing the risk of systemic translocation and photosynthetic toxicity. Overall, internalization with predominant root retention is a common outcome under root exposure scenarios. Root-to-shoot translocation is generally limited, but it can increase when material properties shift through redox transformation or when foliar application bypasses root retention. From a food safety perspective, current evidence remains insufficient to quantify residues in edible organs, particularly fruits and medicinal tissues, across realistic agronomic use patterns. Therefore, applications close to harvest should be avoided until crop-specific residue data and exposure assessments become available. Future work should prioritize traceable labeling approaches and standardized residue analytics across plant tissues and growth stages.

These considerations further reinforce that defining crop and environment-specific safe dose windows and exposure thresholds for GO is a prerequisite for realistic agricultural deployment. For example, cereals grown hydroponically often show no marked inhibition of germination at concentrations ranging from 100 to 200 mg/L. In contrast, concentrations of 1000 mg/L or higher can drive strong root accumulation and induce oxidative stress. Future work should validate such thresholds through long-term field trials and multi-generational assays.

6. Practical Application Guide

Practical use of GO should follow conservative, crop-specific optimization. Promotive or stress-alleviating effects have been reported at low-to-mid mg/L in several systems, whereas exposure at 1000 mg/L or higher under root exposure scenarios is repeatedly associated with strong root accumulation, oxidative stress, and growth inhibition [70,72]. Therefore, the application should start from the lowest effective dose and be validated under the intended matrix and route. Timing should prioritize pre-flowering stages and avoid direct contact with flowers because reproductive processes, particularly pollen-related endpoints, can represent a high sensitivity window [28]. When GO is combined with fertilizers or pesticides, compatibility should be verified because GO has been used as a carrier and in controlled release formulations that can alter the bioavailable fraction and release kinetics of active ingredients [75,76]. From a food safety perspective, evidence remains insufficient to quantify residues across edible organs under realistic agronomic use patterns; thus, applications close to harvest should be avoided until crop-specific residue and exposure data become available [72,74].

7. Conclusions and Future Perspectives

Overall, the effects of GO on plants show clear dependence on both concentration and species. At appropriate doses, GO can improve seed germination and seedling growth and enhance tolerance to salinity, alkalinity, drought, and certain heavy metal stresses, largely through improving soil water retention and water and nutrient transport. Reported activation of antioxidant enzymes and shifts in hormone pathways are frequently consistent with downstream physiological responses and should not be taken as evidence of direct molecular targeting without additional mechanistic validation. At high doses or under unsuitable application conditions, however, GO readily induces strong oxidative stress and membrane damage, suppresses root system architecture formation and the expression of genes involved in nutrient uptake, and subsequently impairs photosynthesis and whole plant growth, which indicates that its use must be accompanied by strict control of dose and exposure scenario.

In the future, GO and its derivatives are expected to play important roles in plant genetic engineering and in the delivery of agricultural inputs. GO has been used to deliver short regulatory RNA (siRNA) into plant tissues and to temporarily switch off target genes in N. tabacum, highlighting GO as a platform for non-integrative (DNA-free) gene regulation and precision delivery, with potential compatibility with CRISPR/Cas ribonucleoprotein cargoes [77]. In addition, the abundant surface functional groups and tunable interfacial chemistry of GO can be exploited to design fertilizer and pesticide formulations that release nutrients or active ingredients in response to environmental cues such as pH and light [75]. Such controlled release systems can improve nutrient and pesticide use efficiency while reducing losses and non-target effects, and therefore offer a material basis for intelligent nano-enabled agrochemicals. For example, GO-reinforced biodegradable coatings have been reported to prolong nutrient release and improve crop biomass, supporting more efficient input use [76]. Combined with the high electrical conductivity of graphene, GO can also be integrated into plant biosensors and flexible implantable electronics to enable in situ monitoring of hormones, stress-related signaling molecules, and ion dynamics, which provides technical support for precision agriculture and the development of a plant Internet of Things. GO-based sensing platforms have also enabled portable detection of stress-related molecular markers, supporting in situ monitoring for precision management [78].

In practical agriculture, humic substances, biochar, and microbial biostimulants can provide partially overlapping benefits with GO via improved water retention, nutrient availability, and rhizosphere function. GO should therefore be positioned as a complementary tool whose value must be demonstrated by head-to-head comparisons against these established inputs, considering both efficacy and environmental fate.

From the perspective of materials synthesis, a recent overview summarized more than 63 plant extracts including Camellia sinensis (L.) Kuntze, A. vera, Eucalyptus, and Panax ginseng that can reduce GO to reduced graphene oxide with C-to-O ratios up to about 22.8, in some cases comparable with hydrazine reduced products, which provides a promising green strategy to produce reduced graphene oxide with tunable physicochemical properties and lower environmental burden for agricultural applications [79]. It is important to note that graphene-based materials in complex biological systems should no longer be regarded as inert carriers. Although these insights come from animal gut systems, they highlight that carbon nanomaterials can be biotransformed by dense microbial communities. Similarly, rhizosphere microbiota and root exudates may mediate GO transformation in plants. Evidence from complex microbiome systems indicates that carbon nanomaterials can undergo microbial biotransformation, which motivates similar attention to rhizosphere and phyllosphere-mediated transformation in plant settings [80,81]. These findings suggest that, in plant systems, more attention should be paid to the transformation of GO by rhizosphere and phyllosphere microorganisms and to the indirect regulation of plant–nanomaterial interactions by these microbial processes. Future research on GO therefore needs, on the one hand, to strengthen multi-scale- and multi-omics-based ecological risk assessment in order to define safe concentration windows and long-term behavior in different environment organism systems and, on the other hand, to draw on concepts from immune reprogramming and microbial ecology to design functionalized GO platforms that combine environmental safety with controllable activity and that support green plant protection, stress resilient cultivation and smart agriculture [82] (Figure 3). Together, GO-enabled gene delivery, precision agrochemical release, in situ biosensing, and microbiome-aware design can converge toward smart and sustainable agriculture and forestry, provided that standardized long-term risk assessment defines safe-dose windows and environmental fate.