The Effects of Graphene-Family Nanomaterials on Plant Growth: A Review

Numerous reports of graphene-family nanomaterials (GFNs) promoting plant growth have opened up a wide range of promising potential applications in agroforestry. However, several toxicity studies have raised growing concerns about the biosafety of GFNs. Although these studies have provided clues about the role of GFNs from different perspectives (such as plant physiology, biochemistry, cytology, and molecular biology), the mechanisms by which GFNs affect plant growth remain poorly understood. In particular, a systematic collection of data regarding differentially expressed genes in response to GFN treatment has not been conducted. We summarize here the fate and biological effects of GFNs in plants. We propose that soil environments may be conducive to the positive effects of GFNs but may be detrimental to the absorption of GFNs. Alterations in plant physiology, biochemistry, cytological structure, and gene expression in response to GFN treatment are discussed. Coincidentally, many changes from the morphological to biochemical scales, which are caused by GFNs treatment, such as affecting root growth, disrupting cell membrane structure, and altering antioxidant systems and hormone concentrations, can all be mapped to gene expression level. This review provides a comprehensive understanding of the effects of GFNs on plant growth to promote their safe and efficient use.


Introduction
Graphene is a two-dimensional nanomaterial composed of one or more layers of carbon atoms with different modifications possible at the edges. Graphene was first discovered by Andre Geim and Constantine Novoselov in 2004 [1], for which they were awarded the Nobel Prize in Physics in 2010. Graphene-family nanomaterials (GFNs) can be divided into several categories based on the number of layers, lateral dimensions, surface chemistry, defect density, and composition; the categories include few-layer graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (RGO), graphene quantum dots (GQD), sulfonated graphene oxide (SGO), amine-functionalized graphene oxide (G-NH 2 ), and hydrated graphene ribbon (HGR) [2,3]. Although members of each group have properties in common, such as a huge specific surface area, excellent mechanical properties, and superior electrical conductivity [4], they also have their own unique properties and functions.
Different GFNs may have varying effects on plant growth due to differences in phy ical and chemical properties. For instance, wheat seedling growth was significantly pro moted by G-NH2 but inhibited by GO treatment [43]. Treatment with HGR rather tha GO or RGO increased seed germination rate, root growth, and resistance to oxidativ stress in wheat [51]. Compared to GO, treatment with lysine@graphene oxide (L-GO) an methionine@graphene oxide (M-GO) dramatically improved pearl millet growth, bio mass, total protein content, photosynthetic pigment content, and yield under salt stre [60]. GO inhibited shoot and root growth at 100 and 250 mg/L, whereas RGO exhibited n obvious toxic morphological effects at the same concentrations [55]. The effects of GFNs on plant growth vary between different species and organs. Most studies have shown that GFN exposure can accelerate seed germination, the first step of plant development, because it promotes water absorption by seeds [9]. GFNs are reported to promote seed germination in spinach [9], chive [9], tomato [30], cotton [41], vinca [42], and wheat [43], but inhibit seed germination in rice [44]. As the organ of direct contact between plants and soil, roots play an important role in plant growth due to their absorption and fixation functions. In the seedling growth stage, GFNs have a large impact on plant root growth, which further affects the growth and development of above-ground organs. The growth of root and aerial organs, which ultimately determines plant biomass, was reportedly stimulated by GFNs in Pinus tabuliformis [39], Aloe vera [45], elm [46], maize [46,47], tomato [30,41], cotton [42], vinca [42], coriander [48], garlic [48], tobacco [49], and wheat [43,50,51] at appropriate concentrations. GFNs also have positive effects on vinca flower number [42], coriander and garlic flower growth [48], tobacco callus growth [49], and watermelon perimeter [52]. In contrast, GFNs inhibition of root and above-ground growth has been reported in species such as cabbage [53], tomato [53], red spinach [53], lettuce [53], rice [44,54,55], wheat [25,43,56], and Brassica napus [57][58][59].
Different GFNs may have varying effects on plant growth due to differences in physical and chemical properties. For instance, wheat seedling growth was significantly promoted by G-NH 2 but inhibited by GO treatment [43]. Treatment with HGR rather than GO or RGO increased seed germination rate, root growth, and resistance to oxidative stress in wheat [51]. Compared to GO, treatment with lysine@graphene oxide (L-GO) and methionine@graphene oxide (M-GO) dramatically improved pearl millet growth, biomass, total protein content, photosynthetic pigment content, and yield under salt stress [60]. GO inhibited shoot and root growth at 100 and 250 mg/L, whereas RGO exhibited no obvious toxic morphological effects at the same concentrations [55]. GO (graphene oxide), GQD (Graphene quantum dots), MS (Murashige and Skoog), FLG (few-layered graphene), RGO (reduced graphene oxide), L-GO (lysine@graphene oxide), M-GO (methionine@graphene oxide), SGO (sulfonated graphene oxide), G-NH 2 (amine-functionalized graphene oxide), HGR (hydrated graphene ribbon), RGR (relative growth rate). NA means the related information is not provided. Similar to most growth-regulating substances, GFNs have concentration-dependent effects on plant growth (whether activating or inhibiting), and therefore an optimal concentration exists for inducing such effects. For example, the optimal concentrations of GO for promoting the growth of Pinus tabuliformis, Aloe vera, and tomato were 25, 50, and 100 mg/L, respectively [39,41,45]. Some studies have demonstrated hormetic biological effects of GFNs, meaning that the response is promoted at low GFN concentrations but inhibited at high concentrations. For example, Ren et al. showed a hormetic response of plant growth to SGO [69]; in another study, 1.5 and 3 µg/L GO increased shoot length and leaf number of Betula pubescens microclones, whereas 15 µg/L reduced shoot height [65].
There is a lag time between application of a substance and the biological effect, and different exposure times often correspond to different development stages. Thus, the biological effects of GFNs often differ with changes in exposure time. For example, plant incubation with graphene (250 -1500 mg/L) for 24 h or 48 h induced no obvious alterations in leaf growth, but exposure for 30 d significantly reduced shoot biomass [70]. Application of 0.1 mg/L graphene to arabidopsis increased leaf area more significantly over 10 d compared to 5 d [57,60]. These results suggest that both short-term and long-term effects of GFNs should be considered when evaluating their biological effects in the following research.
At present, the primary GFN application methods are soil irrigation, addition to solid or liquid medium, and direct injection in vivo. Addition to aqueous solutions or growth media is a widely used strategy in research because the methods are simple, result in a high absorption rate, and include few interference factors. However, an increasing number of experiments have been conducted with soil treatments in recent years because these better mimic natural growth conditions of plants. There have also been some reports of foliar applications and direct injection. Foliar applications of 30 mg/L GO significantly promoted root growth and improved nutritional quality of lettuce [68]. This represents a promising result for GFNs in foliar fertilizer development. Although GO injection into aloe leaves did not directly promote leaf or root growth [45], injection of GO into the watermelon stem significantly increased the fruit perimeter [52]. This is a promising strategy for using GFNs precisely at the lowest possible concentration to minimize environmental release and should therefore be tested with other GFNs and plant species.
GFNs have almost always been reported to show positive effects for plants grown in soil by irrigation and negative effects for plants grown in aqueous solution or medium. We hypothesize that the environment in which plants grow is an important factor affecting the biological effects of GFNs. To test this hypothesis, we grew mung beans in soil, Murashige and Skoog (MS) solid medium, and water solution with 15-200 mg/L GO. The phenotypic analysis revealed that irrigation with GO solution accelerated seed germination and promoted shoot growth for mung beans planted in soil (Figure 2A). In contrast, GO inhibited shoot and root growth in a concentration-dependent manner for mung beans grown in solid MS medium ( Figure 2B). For plants grown in the water solution, GO treatment promoted germination at low concentrations (12.5-50 mg/L) but exhibited obviously toxic effects at higher concentration (>100 mg/L) ( Figure 2C).
The mechanisms underlying the variable biological effects of GFNs on plants grown in different media are unknown. Intuitively, water and mineral nutrients in aqueous and solid media are always abundant and readily available to plants, and GFNs may be less able to play their roles in such an environment. Compared with aqueous solution and solid medium, soil is a more complex and dynamic environment. A number of recent studies may shed light on this phenomenon. Graphene was shown to influence the concentrations of the major ions in soil as follows: sulfate > phosphate > ammonia > nitrate [71]. The oxygencontaining functional groups in GO confer excellent hydrophilicity, and it thus may function to improve the water supply by better collecting and retaining moisture in the soil [9]. As nano-carbons, GFNs may function in enhancing soil aggregation and reducing nutrient loss [41]. In addition, GFNs may affect plant growth by altering soil enzyme activity and microbial community [72]. GFN treatment decreases the activity of some soil enzymes, such as xylosidase, 1,4-β-N-acetyl glucosaminidase, and phosphatase [73], while increasing the activity of others, such as invertase, protease, catalase, and urease [74]. Although in vitro experiments showed that GFN treatment had a toxic effect on the soil microbial community [75], another study found that GO/GN application to the soil increased the diversity of the microbial community [74]. Compared with the mimic aquatic environment, GFN was shown to be much more retained in soil [40,76]. In addition, although high concentrations of GFNs can be toxic to plants, some substances in soil (such as humic acid) are natural antidotes [32]. some soil enzymes, such as xylosidase, 1,4-β-N-acetyl glucosaminidase, and phosphatase [73], while increasing the activity of others, such as invertase, protease, catalase, and urease [74]. Although in vitro experiments showed that GFN treatment had a toxic effect on the soil microbial community [75], another study found that GO/GN application to the soil increased the diversity of the microbial community [74]. Compared with the mimic aquatic environment, GFN was shown to be much more retained in soil [40,76]. In addition, although high concentrations of GFNs can be toxic to plants, some substances in soil (such as humic acid) are natural antidotes [32].

The Physiological and Biochemical Effects of GFNs on Plants
Studies into the effects of GFNs on plant physiology and biochemistry have mainly focused on oxidative stress-related parameters, photosynthesis-related parameters, ion leakage, and water and nutrient content. Parameters related to oxidative stress include accumulation of reactive oxygen species (ROS); activity of antioxidant enzymes, such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT); and levels of oxidized glutathione (GSSG) and malondialdehyde (MDA). Parameters related to photosynthesis include chlorophyll content, photosynthetic rate, transpiration rate, water use efficiency, and stomatal opening.
In general, the negative effects of GFNs reported are increased ROS levels, decreases in antioxidant enzyme activity and photosynthesis-related parameters, increases in MDA and oxidized glutathione, elevated ion leakage, and alterations in moisture and nutrient content [31,53,66,67,77]. In contrast, the positive effects of GFNs tend to be lower ROS levels, enhanced antioxidant enzyme activity and photosynthesis-related parameters, and

The Physiological and Biochemical Effects of GFNs on Plants
Studies into the effects of GFNs on plant physiology and biochemistry have mainly focused on oxidative stress-related parameters, photosynthesis-related parameters, ion leakage, and water and nutrient content. Parameters related to oxidative stress include accumulation of reactive oxygen species (ROS); activity of antioxidant enzymes, such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT); and levels of oxidized glutathione (GSSG) and malondialdehyde (MDA). Parameters related to photosynthesis include chlorophyll content, photosynthetic rate, transpiration rate, water use efficiency, and stomatal opening.
In general, the negative effects of GFNs reported are increased ROS levels, decreases in antioxidant enzyme activity and photosynthesis-related parameters, increases in MDA and oxidized glutathione, elevated ion leakage, and alterations in moisture and nutrient content [31,53,66,67,77]. In contrast, the positive effects of GFNs tend to be lower ROS levels, enhanced antioxidant enzyme activity and photosynthesis-related parameters, and lower MDA content [51,60,63,64]. However, some studies show results that are not consistent with those discussed above. For example, although GO treatment promoted aloe vera growth, MDA levels were increased [45]. This suggests that there may be multiple mechanisms by which GFNs affect plant growth. We have proposed that GO treatment may mimic a weak stress signal, enhancing root growth and the plant's ability to cope with the external environment [39].
Plant hormones, including auxin, cytokinin, abscisic acid (ABA), ethylene, and gibberellin (GA) are important trace substances that regulate plant growth. Changes in levels of these hormones often have a significant effect on the development of plant organs [78]. Studies have shown that positive effects of GO treatment are associated with an increase in IAA levels [41], whereas negative effects are accompanied by an increase in ABA and a decrease in IAA content [58,59].

The Cytological Effects of GFNs on Plants
Several forms of microscopy have been widely used to detect the cytological effects of GFN treatment. Because GFNs have a lamellar structure, cell membranes are vulnerable to mechanical damage by the sharp edges [79,80]. Optical microscopy has shown that stem thickening caused by GO treatment was mainly the result of an increase in cortical cell number [41] and that GO treatment resulted in strong disintegration and loss of epidermal and cortical cells in wheat root [43]. SEM images showed physical damage in wheat root treated with 1000 mg/L GO [81]. In addition, TEM has been used to observe plant ultrastructure, and several such studies have reported a lack of significant changes in cell ultrastructure in response to GFN treatment [9,43]. However, a large number of TEM observations have shown that accumulation of GFNs can lead to destruction of the vacuolar structure and cell membrane, increases in lysosome number, changes in chloroplast shape and the number of starch granules accumulated in chloroplasts, and even serious damage to structures, such as the nucleus [25,27,32,39,77].

Differential Gene Expression in Plants Treated with GFNs
Although the effects of GFNs on morphological, physiological, and cytological phenotypes in plants are well-established, little is known regarding responses at the gene expression level. Analysis of genes affected by GFN treatment not only provides clues for understanding functional mechanisms, but also has significance for effective harnessing of GFNs. First, affected genes could be used as molecular markers to identify the most suitable species and application modes for a given treatment quickly and accurately. Second, identification of affected functional genes would offer the opportunity to genetically engineer plants to mimic the effects of GFN treatment. In recent years, a large number of studies into plant gene expression after GFN treatment have provided opportunities to understand the effects of GFNs at the molecular level. The known differentially expressed genes (DEGs) in response to GFN treatment are shown in Table 3. Table 3. Differentially expressed genes in response to GFNs treatment in plants.

Classification
Species and Tissue Gene Regulation Treatment Ref.
GFNs have been reported to promote the uptake of water and minerals in plants [9,87,88], and we sought to investigate the effects of GFNs on genes encoding membrane transport systems in the literature. Other than the auxin transporter genes mentioned above, nothing else was found. In fact, the expression of auxin transporter genes was regulated by auxin feedback [89], which means the alteration in these genes could be an indirect effect of GFN. We tend to think that the effects of GFNs on root absorption capacity may be independent of the expression of membrane system genes.

Conclusions
Few substances are known to exhibit such varied and antagonistic effects on plants as GFNs, and it is therefore difficult to derive a unified description of GFN mechanisms of action from the current literature. The diversity of effects attributed to GFNs treatment in different studies is caused by differences in plant species, the physicochemical properties of GFNs, concentration, exposure time, and application mode. Among these factors, we tend to think that the physical and chemical properties of GFN may be the most important reason for this contrast. After all, the GFNs used in different studies often come from different sources, and small changes in their modification groups often lead to dramatic changes in its function. The physical and chemical properties of GFNs are easily affected by the substances with which they come into contact. In the future, it should be possible to categorize GFNs by specific functions for uniform use in research and functional applications. We have proposed the creation of GFNs standards as soon as possible. In the absence of a single standard, comparative studies of the biological effects of various types of GFNs under the same conditions may make our conclusions more objective Based on the available literature, it seems likely that GFNs can be absorbed by plant roots and transported to above-ground organs and that GFNs can cross cell membranes and enter organelles. In general, current understanding of the biological effects of GFN treatment is centered on oxidative stress, cell membrane systems, root growth/absorption capacity, and photosynthesis. GFNs may also indirectly affect plant growth by altering soil enzyme activity and microbial communities. Genes with altered expression in response to GFN treatment primarily include growth regulatory factors, transcription factors, hormonerelated genes, nutrition metabolism-related genes, stress-related genes, and those encoding chloroplast proteins. However, the roles of these genes in responding to GFN treatment require further study.