Skip to main content

A Novel Mechanism Underlying Multi-walled Carbon Nanotube-Triggered Tomato Lateral Root Formation: the Involvement of Nitric Oxide


Abundant studies revealed that multi-walled carbon nanotubes (MWCNTs) are toxic to plants. However, whether or how MWCNTs influence lateral root (LR) formation, which is an important component of the adaptability of the root system to various environmental cues, remains controversial. In this report, we found that MWCNTs could enter into tomato seedling roots. The administration with MWCNTs promoted tomato LR formation in an approximately dose-dependent fashion. Endogenous nitric oxide (NO) production was triggered by MWCNTs, confirmed by Greiss reagent method, electron paramagnetic resonance (EPR), and laser scanning confocal microscopy (LSCM), together with the scavenger of NO. A cause-effect relationship exists between MWCNTs and NO in the induction of LR development, since MWCNT-triggered NO synthesis and LR formation were obviously blocked by the removal of endogenous NO with its scavenger. The activity of NO generating enzyme nitrate reductase (NR) was increased in response to MWCNTs. Tungstate inhibition of NR not only impaired NO production, but also abolished LR formation triggered by MWCNTs. The addition of NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of mammalian nitric oxide synthase (NOS)-like enzyme, failed to influence LR formation. Collectively, we proposed that NO might act as a downstream signaling molecule in MWCNT control of LR development, at least partially via NR.


There have many biological and biomedical applications of carbon nanotubes [1, 2]. Due to the unique ability to easily penetrate cell membranes, the biosafety of carbon nanotubes is always a debate topic [3, 4]. Meanwhile, since the production and use of carbon nanotubes grow rapidly, it becomes important to characterize the detailed mechanisms of its cytotoxicity in human beings and mammalians, and recently in plants [3,4,5,6,7,8,9]. It is well-known that plants and their communities are very important for humans and environment, and they are at risk of carbon nanotubes exposure either, due to buildup in soils through biosolid fertilizer application [6, 10, 11]. As the important members of carbon nanotubes, the toxicity of multi-walled carbon nanotubes (MWCNTs), consisting of multiple rolled layers of graphene, has been widely investigated. Studies in mammalian revealed that the exposure with both MWCNTs and single-walled carbon nanotube induced oxidative damage and NF-κB activation in human keratinocytes and A549 cells [9, 12]. MWCNTs and single-walled carbon nanotube can fuse with the plasma membrane, thus causing cell damage through lipid peroxidation and oxidative stress [9, 11, 13, 14]. Cytotoxicity and oxidative stress triggered by MWCNTs, as well as modest inflammatory responses, were observed in human umbilical vein endothelial cells [15]. Previous study suggested that the primary toxicity of MWCNTs in red spinach was mainly derived from reactive oxygen species (ROS) overproduction, and the toxic effects could be reversed by the supplemented ascorbic acid [7]. In this sense, MWCNTs is considered as a new stressed factor to organisms, either in animals or in plants.

Lateral root (LR) formation, an important determinant of root architecture, has been considered as an indicator of adaptive response to various stresses [16]. In higher plants, the formation of LR is influenced by phytohormones and a wide range of environmental cues, including water availability, nutrients, and abiotic stress, such as hypoxia and heavy metal stress [17,18,19]. Meanwhile, ample evidence confirmed that the formation of LR not only acts as a physical support, but also contributes to water and nutrient uptake for plant growth and development [19,20,21]. Different environmental clues could trigger several specific stress-induced morphogenic response (SIMR) phenotypes, including the promotion of LR formation and an inhibition of root elongation [17].. The regulation of LR formation is also tightly controlled by phytohormones, such as auxin, and the activation of cell cycle regulatory genes in response to auxin was suggested [19, 22]. Meanwhile, the involvement of some small molecules in auxin-triggered root organogenesis was confirmed in cucumber, tomato, soybean, and rapeseed plants [23,24,25,26,27]. These small molecules include hydrogen peroxide (H2O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen gas (H2).

Among these, NO, a free radical gas, has been shown to have multiple physiological functions in plants [28, 29]. Besides the enhancement of plant adaptation against stresses, the functions of NO include the promotion of root hair development, adventitious rooting, and lateral root formation [30,31,32,33], although the enzymatic resource(s) of NO biosynthesis in those aforesaid processes remains elusive. In animals, the synthesis of NO from l-arginine is catalyzed by the heme-containing enzyme nitric oxide synthase (NOS) [34]. Although gene(s) encoding NOS enzymes has not been identified in plants, the mammalian NOS-like activity is detected widely [35, 36], and the inhibitors of mammalian NOS, such as NG-nitro-l-arginine methyl ester hydrochloride (l-NAME), can inhibit NO generation in plants [25, 33, 36,37,38,39]. Importantly, ample genetic evidence revealed that NO can be produced by nitrate reductase (NR), a well-known enzyme responsible for nitrogen metabolism in plants [28]. The involvement of NR-mediated NO production in stomatal closure and cold acclimation has been demonstrated genetically [37, 38]. Our previous study showed that NR-dependent NO synthesis is involved in auxin-induced hydrogen gas-mediated lateral root formation [39].

Until now, different responses in LR formation, promotion or inhibition, were respectively reported in various plant species when supplemented with nanomaterials, including MWCNTs [40,41,42,43], gold nanoparticles (Au NP, [44]), zinc oxide nanoparticles (ZnO NP [45, 46];), titanium dioxide nanoparticles (TiO2 NP [46];), and graphene oxide (GO [47,48,49];) (Table 1), and no study has yet provided definitive proof of a role of NO in above responses. In this study, the detection of endogenous NO by Greiss reagent method, laser scanning confocal microscopy (LSCM), and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in MWCNT-treated tomato seedlings. Afterwards, LR formation was observed. We further study the involvement of NO in LR formation triggered by MWCNTs, by manipulating endogenous NO levels using NO scavenger and antagonists that inhibit NR and mammalian-like NOS activity. Further experiment revealed that NR-dependent NO might be, at least partially, essential for LR formation in response to MWCNTs. This work thus opens a new window for understanding the biological effects of nanomaterials in plants.

Table 1 Different responses in LR formation triggered by nanomaterials

Materials and Methods


Unless stated otherwise, all the other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). MWCNTs, purchased from Sigma-Aldrich, was characterized as previously described [50]. The outer diameter, inter diameter, and the length of MWCNTs were 6–12 nm, 2.5–5 nm, and 1–9 μm, respectively. After sonication treatment, the obtained homogenate colloidal suspension was sterilized and used.

Other carbon nanoparticles were obtained from Nanjing XFNANO Materials Tech Co., Ltd., including single-walled carbon nanotubes (SWCNTs, XFS22; purity > 95%, diameter 1–2 nm, length 5–30 μm, special surface area > 1075 m2/g), graphene (XF001W; purity ~ 99%, diameter 0.5–5 μm, thickness ~ 0.8 nm, single layer ratio ~ 80%, BET surface area 500~1000 m2/g; electrical resistivity  0.30 Ω.cm), and active carbon (AC, XFP06; purity > 95%, particle size 5 ± 1 μm, pore volume 1–1.2 cm3/g, aperture 2.0–2.2 nm, special surface area ~ 1500–1700 m2/g).

Additionally, sodium nitroprusside (SNP) was used as a NO-releasing compound [30,31,32,33]. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) was regarded as a scavenger of NO [51,52,53,54]. Tungstate (Tg; an inhibitor of NR [28, 33, 37, 55,56,57];) and NG-nitro-L-arginine methyl ester hydrochloride (NAME; an inhibitor of mammalian NOS-like enzyme [25, 33, 36,37,38,39];) were also applied. In this study, the concentrations of above chemicals were determined in the pilot experiments, from which the significant responses were observed.

Plant Material and Growth Conditions and Determination of LR Formation

Tomato (Solanum lycopersicum L.) seeds “Jiangshu 14” were kindly supplied by Jiangsu Agricultural Institutes, Nanjing, Jiangsu Province, China. Selected seeds of identical size were germinated in distilled water at 25 ± 1 °C in the dark for 3 days. The selected identical seedlings with radicles 2–3 mm were then transferred to 6 mL treatment solutions containing the indicated concentrations of MWCNTs, 200 nM 1-naphthylacetic acid (NAA; a well-known auxin), 0.1 mM SNP, 0.2 mM cPTIO, 20 μM tungstate (Tg), 0.2 mM NAME, and other carbon nanoparticles, including 5 mg/mL single-walled carbon nanotubes (SWCNTs), graphene, and active carbon (AC), alone or in combination for the indicated time points. Seedlings were grown in an illuminating incubator (25 ± 1 °C) with a light intensity of 200 μmol m−2 s−1 at 14/10 h (light/dark) photoperiod.

After treatments, pictures were taken, and the number and length of emerged lateral root (> 1 mm) per seedling were then determined by using the Image J software ( [39, 58]. As described previously, only the lateral root-inducible segments were used for the subsequent analysis.

Imaging of MWCNT Distribution by Transmission Electron Microscopy

The distribution of MWCNTs in tomato seedling root was characterized using the transmission electron microscopy (TEM; JEOL, JEM-200CX, Tokyo, Japan). Sample preparation for TEM analysis was according to the previous protocol [59].

Imaging of Endogenous NO by Laser Scanning Confocal Microscope

NO imaging was carried out by using a fairly specific NO fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA). After the probe was thoroughly washed, the images were obtained using the Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany, excitation at 488 nm, emission at 500–530 nm for NO analysis). In our experiment, 20 individual samples were randomly selected and measured per treatment. Photographs are representative of identical results.

NO Content Determined by Griess Reagent Assay

According to the methods previously described [50], NO content was determined with the Griess reagent assay. Importantly, for escaping the interfering caused by the concentrated nitrate and nitrite contents in plants, the identical samples preincubated in 200 μM cPTIO (the scavenger of NO) for 30 min were regarded as the blank samples. After the addition of Griess reagent for 30 min, absorbance was recorded at 540 nm, and NO content was determined by comparison to a standard curve of NaNO2.

Determination of NO with Electron Paramagnetic Resonance (EPR)

According to our previous methods [39, 55, 60], the determination of NO level using electron paramagnetic resonance (EPR) was carried out. The organic solvent layer was used to determine NO on a Bruker A300 spectrometer (Bruker Instrument, Karlsruhe, Germany) under the following conditions: room temperature; microwave frequency, 9.85 GHz; microwave power, 63.49 mW; and modulation frequency, 100.00 kHz.

Determination of Nitrate Reductase (NR) Activity

The NR activity was detected spectrophotometrically at 540 nm according to the previous method [57]. The produced nitrite was determined spectrophotometrically at 540 nm by the addition of 1 mL of 1% (w/v) sulfenilamide in 3 M HCl together with 1 mL of 0.02% (v/v) n-(1-naphthyl)-ethylenediamine.

Statistical Analysis

Where indicated, results were expressed as the mean values ± SE of three independent experiments with three biological replicates for each. Statistical analysis was performed using the SPSS Statistics 17.0 software. For statistical analysis, Duncan’s multiple test (p < 0.05) was chosen as appropriate.


MWCNTs not only Entry into Root Cells, but also Promote LR Formation

LR formation is a major determinant of root systems architecture. To investigate the effect of MWCNTs on LR formation, 3-day-old tomato seedlings were incubated with a range of concentrations of MWCNTs (0.05, 0.5, 5, and 50 mg/mL) for 3 days. The application of 1-naphthylacetic acid (NAA) was regarded as a positive control. In our experiment, both LR number and length were determined as two parameters of LR formation. As shown in Fig. 1, compared to the control samples, exogenous MWCNTs significantly induced LR formation in an approximately dose-dependent manner, with a maximal effect in 5 and 50 mg/mL. Similar inducible response was observed when 200 nM NAA was administrated. Considering the cost of MWCNTs and inducible response in LR formation, 5 mg/mL MWCNTs was applied in the following experiments.

Fig. 1
figure 1

MWCNT-induced tomato LR formation was in an approximately dose-dependent manner. Three-day-old tomato seedlings were treated with 200 nM NAA and the indicated concentrations of MWCNTs, respectively. The number and length of emerged lateral root (> 1 mm) per seedling were then determined after 3 days of treatment. There were 30 (10 × 3) plants in three biological replicates, and the experiments were conducted for 3 times. Data are the means ± SE. Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple test

To validate the specific function of MWCNTs in the induction of LR formation, we further investigate whether the other allotropies of MWCNTs also have such inducible effects. As shown in Fig. 2a, all these carbon nanomaterials exhibited toxic effects on shoot growth (data not shown). Interestingly, the application of MWCNTs, single-walled carbon nanotubes (SWCNTs), graphene, and active carbon with identical concentration (5 mg/mL) could differentially result in the increases in LR number and length, compared to the chemical-free control plants (Fig. 2b). Among these chemicals, the maximal inducible response was discovered in MWCNT-incubated tomato seedlings.

Fig. 2
figure 2

Changes in lateral root formation in response to different carbon nanoparticles. Three-day-old tomato seedlings were treated with distilled water (Con), 5 mg/mL MWCNT, single-walled carbon nanotubes (SWCNTs), graphene, and active carbon (AC), respectively, for another 3 days. a Representative photos were then taken. b The number and length of emerged lateral root (> 1 mm) per seedling were then determined as well. Scale bar = 50 mm. There were 30 (10 × 3) plants in three biological replicates, and the experiments were conducted for 3 times. Data are the means ± SE. Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple test

By the aid of transmission electron microscopy (TEM), the distribution of MWCNTs can be evaluated easily. The results shown in Fig. 3 revealed that MWCNTs, when exogenously applied, could be absorbed by tomato seedlings, and the distribution of MWCNTs was found to be in root cells. This result can be understood, since seedling roots are directly cultured in liquid solution containing MWCNTs.

Fig. 3
figure 3

Distribution of MWCNTs in tomato roots. TEM images of 3-day-old tomato seedlings treated with distilled water (Con; a) or 5 mg/mL MWCNTs (b) for 1 day were taken. Red arrow indicates cell wall, while white arrow indicates MWCNTs

MWCNT-Induced NO Synthesis and Thereafter LR Formation Were Sensitive to cPTIO, a Scavenger of NO

To investigate whether NO is also involved in MWCNT-induced LR formation, the function of NO in LR formation elicited by MWCNTs was assessed by manipulating endogenous NO levels using NO-releasing compound and the scavenger. Similar to the previous results [31], the administration of sodium nitroprusside (SNP) could result in the induction of LR formation, and an additive response was observed when SNP and MWCNTs were applied together (Fig. 4). When 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO; a scavenger of NO) was added, the promotion responses in LR formation caused by MWCNTs were significantly impaired. Alone, cPTIO could inhibit LR development, compared to the chemical-free control, indicating the important role of endogenous NO in root organogenesis.

Fig. 4
figure 4

MWCNT-induced LR formation was sensitive to the removal of endogenous NO with cPTIO, its scavenger. Three-day-old tomato seedlings were treated with distilled water, 5 mg/mL MWCNT, 0.1 mM SNP, 0.2 mM cPTIO, alone or in combination for 3 days. Afterwards, the number and length of emerged lateral root (> 1 mm) per seedling were then determined. There were 30 (10 × 3) plants in three biological replicates, and the experiments were conducted for 3 times. Data are the means ± SE. Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple test

In order to further evaluate the important role of endogenous NO in MWCNT response, a time course of NO production in vivo was firstly detected with Greiss reagent method. During above determination, the identical filtrate pretreated with cPTIO was regarded as a blank for the accurate results. It was observed that NO production in tomato seedling roots was increased dramatically till 24 h after MWCNT treatment and then recovers to the initial levels (48 h; Fig. 5a). Above maximal level of endogenous NO triggered by MWCNTs for 24 h was obviously abolished by cPTIO, a scavenger of NO, suggesting the specific role of NO.

Fig. 5
figure 5

MWCNT-induced NO production was blocked by cPTIO, the scavenger of NO. Three-day-old tomato seedlings were treated with distilled water and 5 mg/mL MWCNTs with or without 0.2 mM cPTIO, respectively. a Changes in NR activity (left), and NO production (right) determined using Greiss reagent method. b After treamtent for 24 h, the NO signal was analyzed by LSCM (left) and EPR (right). Scale bar = 0.1 mm. Data are the means ± SE. Bars denoted by the same letter did not differ significantly at p < 0.05 level according to Ducan’s multiple test

To confirm above results, both LSCM and ESR were adopted. Firstly, the changes in endogenous NO levels in seedling roots of tomato were monitored by labeling NO using the cell-permeable, fairly NO-specific fluorescent probe DAF-FM DA and imaging with LSCM. Similar to the previous results (Fig. 5a), in the presence of cPTIO, the increased DAF-FM-dependent fluorescence intensity triggered by MWCNTs was greatly abolished (Fig. 5b). These results implied that the DAF-FM-triggered fluorescence is related to endogenous NO levels in tomato seedling roots.

MWCNT-induced NO production was confirmed by EPR spectroscopy. As expected, seedling roots treated for 24 h with MWCNTs presented the typical hyperfine structure triplet of the NO complex. However, the addition of cPTIO abolished above signal, indicating that MWCNT exposure did result in a strong NO production (Fig. 5b). Collectively, these data suggested that NO synthesis might be required for MWCNT-triggered LR formation in tomato seedlings.

NR Might Be Responsible for MWCNT-Induced NO Production and Thereafter LR Formation

Since NR and mammalian-like NOS are two major enzymes related to NO synthesis in plants, both tungstate (a NR inhibitor) and NAME (a mammalian NOS inhibitor) were applied in the subsequent experiment. Here, tungstate treatment substantially suppressed the promotion of LR formation in MWCNT-treated tomato seedling roots (Fig. 6). Comparatively, the induction of LR formation triggered by MWCNTs was not strongly inhibited by the addition of NAME, indicating that mammalian-like NOS might be not the target NO synthetic enzyme responsible for NO production elicited by MWCNTs. It was also observed that a slight but no significant decrease in LR formation was observed in tomato seedlings when either tungstate or NAME was separately applied.

Fig. 6
figure 6

Changes in LR formation in response to MWCNTs and two inhibitor of NO synthesis. Three-day-old tomato seedlings were treated with 5 mg/mL MWCNT, 20 μM tungstate (Tg), 0.2 mM NAME, alone or in combination for 3 days. Afterwards, the number and length of emerged lateral root (> 1 mm) per seedling were then determined. There were 30 (10 × 3) plants in three biological replicates, and the experiments were conducted for 3 times. Data are the means ± SE. Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple test

The role of NR in MWCNT-triggered LR formation was further examined by monitoring NO production in response to applied MWCNTs with or without tungstate. Compared to the changes in endogenous NO production (Fig. 5a), time-course analysis in NR activity showed the similar tendency (Fig. 7a), also peaking at 24 h after treatment with MWCNTs. These results suggested that MWCNT-induced increase in NO production may mainly result from enhanced activity of NR. Consistently, the inhibition of NR-dependent NO production by tungstate was confirmed by using Greiss reagent method (Fig. 7b), LSCM, and EPR (Additional file 1: Figure S1).

Fig. 7
figure 7

MWCNT-induced NO production was blocked by tungstate, an inhibitor of NR. Three-day-old tomato seedlings were treated with distilled water and 5 mg/mL MWCNTs with or without 20 μM tungstate (Tg). Changes in NR activity (a) and NO production (b) determined using Greiss reagent method. Data are the means ± SE. Bars denoted by the same letter did not differ significantly at p < 0.05 level according to Ducan’s multiple test


Phytotoxicity is a significant consideration in understanding the potential environmental impact of nanoparticles [4, 7, 61,62,63]. Abundant evidence revealed that MWCNTs are toxic to plants, including inducing oxidative damage, inhibiting seed germination, root growth, and development [11, 63, 64]. However, being as a phenotype of SIMR, root branching through lateral root formation is an important component of the adaptability of the root system to various environmental cues [17]. In this work, we integrated biological, pharmacological, and biochemical analysis to show the involvement of NR-mediated NO production in MWCNT-induced LR formation, at least partially in our experimental conditions. Also, the function of NO in root organogenesis stimulated by MWCNTs emphasized the central roles of this second messenger involved in plant developmental process and adaption against stress [29,30,31,32,33, 37, 38].

First, we confirmed that 5 mg/mL MWCNTs (OD 6–12 nm) could enter into root tissues (Fig. 3). Afterwards, the induction of tomato LR formation was observed (Fig. 1), mimicking the induction roles of NAA and SNP (Fig. 4), a well-known NO-releasing compound [30, 31]. Similar inducing responses were discovered in resinous trees [41], lettuce [42], and Arabidopsis [43] when challenged with MWCNTs (OD 6–13 nm, about 9.5 nm, and 30–40 nm, respectively). For example, the application with either pristine MWCNT (p-MWCNT) or carboxyl-functionalized MWCNT (c-MWCNT) (average diameter 9.5 nm) could promote the development of LR in lettuce seedlings [42]. By contrast, the inhibition of primary root and even LR formation were simultaneously found in soybean plants when subjected to MWCNTs (OD 20–70 nm [40];). By comparing with the data in outer diameter of MWCNTs (Table 1), we supposed that MWCNT-exhibited effects on LR formation varied with their diameters, showing the promotion with lower diameter and the inhibition with higher diameter. Certainly, related mechanism should be carefully investigated. Similar phenomenon was confirmed in plant salinity tolerance [50]. Combined with above results, it was further deduced that the function of nanomaterials may vary from species, and vice versa, different types of nanomaterials may cause various biological effects. However, other influencing factors, such as different doses of MWCNTs [48] and even plant growth conditions, could be not easily ruled out.

Compared with other nanomaterials, including SWCNT, graphene, and AC with an identical concentration, the maximal induction in LR formation and even toxic effects on shoot growth were observed in MWCNTs (Fig. 2). These might be related to the special physical characteristics of MWCNTs, one type of nanomaterials that have high electrical conductivity, large specific surface area, high aspect ratio, and remarkable thermal stability [65]. The toxic effects of nanomaterials have been widely reported in cucumber, cabbage, carrot, onion etc. [66, 67].

Ample evidence showed that NO, acting as a signaling molecule, can regulate a wide range of plant processes from environmental adaptation to development and the latter of which includes seed germination and root organogenesis [29, 68,69,70,71,72,73]. Our subsequent experiment revealed that NO may be involved in MWCNT-induced LR formation. Although several methods for imaging NO production in plant cells have been applied, the disadvantages, including the lack of sensitivity and the interference by NO-independent molecules, may exist in each method [74]. Thus, three methods responsible for NO imaging and determination, including Greiss reagent method, LSCM, and EPR, together with the application of cPTIO, a scavenger of NO, were applied in our experimental conditions. By using three methods, we observed that an increased endogenous NO production induced by MWCNTs in tomato seedlings was abolished by cPTIO (Fig. 5), a scavenger of NO [30,31,32]. Importantly, this process was correlated to the biological response of MWCNT-induced LR development, which was severely blocked when cPTIO was applied simultaneously (Fig. 4).

Further evaluation of these responses and the potential source(s) of NO induced by exogenously applied MWCNTs revealed that NO production and thereafter LR formation could be attributed to NR activity. In plants, NO production mainly generates from NR and mammalian NOS-like protein [28]. However, plant NOS gene is still not identified [35, 75, 76], although some experiments using the inhibitors of the mammalian NOS enzyme provided some evidence of l-arginine-dependent pathway in NO production [36, 76]. NR is confirmed to be the most important sources of NO in plants [28]. Previous studies showed that NR-dependent NO production functions as a nitrate-related signal involved in the regulation of root architecture [32, 33]. Besides, NR-dependent NO production was closely associated with in cold acclimation [38], salinity tolerance [50], and abscisic acid-induced stomatal closure [77]. Our results further revealed that tungstate (an inhibitor of NR) obviously impaired MWCNT-induced LR formation, especially in LR length (Fig. 6). By contrast, there was only a slight decrease in LR length, and no significant difference observed in LR number when l-NAME (an inhibitor of mammalian NOS) was used. Consistently, biochemical assay showed that NR activity was increased obviously by MWCNTs (Fig. 7a), paralleled to the changes in NO production (Fig. 5a). Above responses could be totally blocked by tungstate (Fig. 7a, Additional file 1: Figure S1). We thus deduced that the increased endogenous NO production induced by MWCNTs was mainly attribute to NR pathway. Certainly, further genetic evidence should be investigated.


In summary, we provide evidence to show that MWCNT-induced NO production via NR might be required for tomato lateral root formation and this was summarized in Fig. 8. Importantly, above findings provide insights into the intricate molecular mechanism of MWCNTs functions in plants.

Fig. 8
figure 8

Schematic representation of the proposed MWCNT-induced tomato lateral root formation mainly via NR-dependent NO production. The role of mammalian NOS-like enzyme was preliminarily ruled out

Availability of Data and Materials

All data are fully available without restriction.



Carbon monoxide


2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt


4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate


Electron paramagnetic resonance


Graphene oxide

H2 :

Hydrogen gas

H2O2 :

Hydrogen peroxide


NG-Nitro-l-arginine methyl ester


Lateral root


Laser scanning confocal microscopy


Multi-walled carbon nanotubes


1-Naphthylacetic acid


Nitric oxide


Nitric oxide synthase


Nitrate reductase


Reactive oxygen species


Stress-induced morphogenic response


Sodium nitroprusside


Single-walled carbon nanotubes


Transmission electron microscopy




  1. Bianco A, Prato M (2003) Can carbon nanotubes be considered useful tools for biological applications? Adv Mater 15:1765–1766

    CAS  Article  Google Scholar 

  2. Baughman RH, Zakhidov AA, Heer DA (2002) Carbon nanotubes-the route toward applications. Science 297:787–792

    CAS  Article  Google Scholar 

  3. Liu Q, Chen B, Wang Q, Shi X, Xiao Z, Lin J, Fang X (2009) Carbon nanotubes as molecular transporters for walled plant cells. Nano Lett 9:1007–1010

    CAS  Article  Google Scholar 

  4. Lam CW, James JT, Mccluskey R, Arepalli S, Hunter RL (2006) A review of carbon nanotube toxicity and assessment of potential occupational and environmental health risks. Crit Rev Toxicol 36:189–217

    CAS  Article  Google Scholar 

  5. Servin A, Elmer W, Mukherjee A, la Torre-Roche RD, Hamdi H, White JC, Bindraban P, Dimkpa C (2015) A review of the use of engineered nanomaterials to suppress plant disease and enhance crop yield. J Nanopart Res 17:92

    Article  CAS  Google Scholar 

  6. Yan S (2013) Single-walled carbon nanotubes selectively influence maize root tissue development accompanied by the change in the related gene expression. J Hazard Mater 246-247:110–118

    CAS  Article  Google Scholar 

  7. Begum P, Fugetsu B (2012) Phytotoxicity of multi-walled carbon nanotubes on red spinach (Amaranthus Tricolor L.) and the role of ascorbic acid as an antioxidant. J Hazard Mater 243:212–222

    CAS  Article  Google Scholar 

  8. Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann MC (2005) In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicol Sci 88:412–419

    CAS  Article  Google Scholar 

  9. Manna SK, Sarkar S, Barr J, Wise K, Barrera EV, Jejelowo O, Rice-Ficht AC, Ramesh GT (2005) Single-walled carbon nanotube induces oxidative stress and activates nuclear transcription factor-κB in human keratinocytes. Nano Lett 5:1676–1684

    CAS  Article  Google Scholar 

  10. Oleszczuk P, Jośko I, Xing B (2011) The toxicity to plants of the sewage sludges containing multiwalled carbon nanotubes. J Hazard Mater 186:436–442

    CAS  Article  Google Scholar 

  11. Tan X, Lin C, Fugetsu B (2009) Studies on toxicity of multi-walled carbon nanotubes on suspension rice cells. Carbon 47:3479–3487

    CAS  Article  Google Scholar 

  12. Ye S, Wu Y, Hou Z, Zhang Q (2009) ROS and NF-κB are involved in upregulation of IL-8 in A549 cells exposed to multi-walled carbon nanotubes. Biochem Bioph Res Co 379:643–648

    CAS  Article  Google Scholar 

  13. Cui D, Tian F, Ozkan CS, Wang M, Gao H (2005) Effect of single wall carbon nanotubes on human HEK293 cells. Toxicol Lett 155:73–85

    CAS  Article  Google Scholar 

  14. Ghorbanpour M, Hadian J (2015) Multi-walled carbon nanotubes stimulate callus induction, secondary metabolites biosynthesis and antioxidant capacity in medicinal plant Satureja khuzestanica grown in vitro. Carbon 94:749–759

    CAS  Article  Google Scholar 

  15. Long J, Li X, Kang Y, Ding Y, Gu Z, Cao Y (2018) Internalization, cytotoxicity, oxidative stress and inflammation of multi-walled carbon nanotubes in human endothelial cells: influence of pre-incubation with bovine serum albumin. RSC Adv 8:9253–9260

    CAS  Article  Google Scholar 

  16. Bellini C, Pacurar D, Perrone I (2014) Adventitious roots and lateral roots: similarities and difference. Annu Rev Plant Biol 65:639–666

    CAS  Article  Google Scholar 

  17. Potters G, Pasternak TP, Guisez Y, Palme KJ, Jansen MAK (2007) Stress-induced morphogenic responses: growing out of trouble? Trends Plant Sci 12:98–105

    CAS  Article  Google Scholar 

  18. Péret B, Rybe B, Casimiro I, Benková E, Swarup R, Laplaze L, Beeckman T, Bennett MJ (2009) Arabidopsis lateral root development: an emerging story. Trends Plant Sci 14:399–408

    Article  CAS  Google Scholar 

  19. Lavenus J, Goh T, Roberts I, Guyomarch S, Lucas M, Smet ID (2013) Lateral root development in Arabidopsis: fifty shades of auxin. Trends Plant Sci 18:450–458

    CAS  Article  Google Scholar 

  20. Casimiro I, Beeckman T, Graham N, Bhalerao R, Zhang H, Casero P, Sandberg G, Bennett MJ (2003) Dissecting Arabidopsis lateral root development. Trends Plant Sci 8:165–171

    CAS  Article  Google Scholar 

  21. Benková E, Bielach A (2010) Lateral root organogenesis-from cell to organ. Curr Opin Plant Biol 13:13677–13683

    Article  Google Scholar 

  22. del Pozo JC, Lopez-Matas MA, Ramirez-Parra E, Gutierrez C (2005) Hormonal control of the plant cell cycle. Physiol Plant 123:173–183

    Article  CAS  Google Scholar 

  23. Correa-Aragunde N, Graziano M, Chevalier C, Lamattina L (2006) Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato. J Exp Bot 57:581–588

    CAS  Article  Google Scholar 

  24. Su G, Zhang W, Liu Y (2006) Involvement of hydrogen peroxide generated by polyamine oxidative degradation in the development of lateral roots in soybean. J Integr Plant Biol 48:426–432

    CAS  Article  Google Scholar 

  25. Cao Z, Xuan W, Liu Z, Li X, Zhao N, Xu P, Wang Z, Guan R, Shen W (2007) Carbon monoxide promotes lateral root formation in rapeseed. J Integr Plant Biol 49:1070–1079

    CAS  Article  Google Scholar 

  26. Cao Z, Fang T, Chen M, Li J, Shen W, Huang L (2014) Involvement of haem oxygenase-1 in hydrogen peroxide-induced lateral root formation in tomato. Acta Physiol Plant 36:931–943

    CAS  Article  Google Scholar 

  27. Lin Y, Zhang W, Qi F, Cui W, Xie Y, Shen W (2014) Hydrogen-rich water regulates cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner. J Plant Physiol 171:1–8

    CAS  Article  Google Scholar 

  28. Rockel P, Strube F, Rockel A, Wildt J, Kaiser WM (2002) Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. J Exp Bot 53:103–110

    CAS  Article  Google Scholar 

  29. Fancy NN, Bahlmann AK, Loake GJ (2016) Nitric oxide function in plant abiotic stress. Plant Cell Environ 40:462–472

    Article  CAS  Google Scholar 

  30. Pagnussat GC, Simontacchi M, Puntarulo S, Lamattina L (2002) Nitric oxide is required for root organogenesis. Plant Physiol 129:954–956

    CAS  Article  Google Scholar 

  31. Correa-Aragunde N, Graziano M, Lamattina L (2004) Nitric oxide plays a central role in determining lateral root development in tomato. Planta 218:900–905

    CAS  Article  Google Scholar 

  32. Lombardo MC, Graziano M, Polacco JC, Lamattina L (2006) Nitric oxide functions as a positive regulator of root hair development. Plant Signal Behav 1:28–33

    Article  Google Scholar 

  33. Qi F, Xiang Z, Kou N, Cui W, Xu D, Wang R, Zhu D, Shen W (2017) Nitric oxide is involved in methane-induced adventitious root formation in cucumber. Physiol Plant 159:366–377

    CAS  Article  Google Scholar 

  34. Li H, Poulos TL (2005) Structure-function studies on nitric oxide synthases. J Inorg Biochem 99:293–305

    CAS  Article  Google Scholar 

  35. Zemojtel T, Fröhlich A, Palmieri MC, Kolanczyk M, Mikula I, Wyrwicz LS, Wanker EE, Mundols S, Vingron M, Martasek P, Durner J (2006) Plant nitric oxide synthase: a never-ending story? Trends Plant Sci 11:524–525

    CAS  Article  Google Scholar 

  36. Hao G, Xing Y, Zhang J (2008) Role of nitric oxide dependence on nitric oxide synthase-like activity in the water stress signaling of maize seedling. J Inorg Biochem 50:435–442

    CAS  Google Scholar 

  37. Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45:113–122

    CAS  Article  Google Scholar 

  38. Zhao M, Chen L, Zhang L, Zhang W (2009) Nitric reductase dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151:755–767

    CAS  Article  Google Scholar 

  39. Cao Z, Duan X, Yao P, Cui W, Cheng D, Zhang J, Jin Q, Chen J, Dai T, Shen W (2017) Hydrogen gas is involved in auxin-induced lateral root formation by modulating nitric oxide synthesis. Int J Mol Sci 18:2084

    Article  CAS  Google Scholar 

  40. Zaytseva O, Wang Z, Neumann G (2017) Phytotoxicity of carbon nanotubes in soybean as determined by interactions with micronutrients. J Nanopart Res 19:29

    Article  CAS  Google Scholar 

  41. Lara-Romero J, Campos-García J, Dasgupta-Schubert N, Borjas-García S, Tiwari DK, Paraguay-Delgado F, Jiménez-Sandoval S, Alonso-Nuñez G, Gómez-Romero M, Lindig-Cisneros R, De la Cruz HR, Villegas JA (2017) Biological effects of carbon nanotubes generated in forest wildfire ecosystems rich in resinous trees on native plants. PeerJ 5:3658

    Article  CAS  Google Scholar 

  42. Das KK, You Y, Torres M, Barrios-Masias F, Wang XL, Tao S, Xing BS, Yang Y (2018) Development and application of a digestion-Raman analysis approach for studying multiwall carbon nanotube uptake in lettuce. Environ Sci-Nano 5:659–668

    CAS  Article  Google Scholar 

  43. Fan X, Xu J, Lavoie M, Peijnenburg WJGM, Zhu YC, Lu T, Fu Z, Zhu T, Qian H (2018) Multiwall carbon nanotubes modulate paraquat toxicity in Arabidopsis thaliana. Environ Pollut 233:633–641

    CAS  Article  Google Scholar 

  44. Gopinath K, Gowri S, Karthika V, Arumugam A (2014) Green synthesis of gold nanoparticles from fruit extract of Terminalia arjuna, for the enhanced seed germination activity of Gloriosa superba. J Nanostruct Chem 4:115

    Article  Google Scholar 

  45. Watson JL, Fang T, Dimkpa CO, Britt DW, Mclean JE, Jacobson A, Anderson AJ (2015) The phytotoxicity of ZnO nanoparticles on wheat varies with soil properties. Biometals 28:101–112

    CAS  Article  Google Scholar 

  46. Hajra A, Mondal NK (2017) Effects of ZnO and TiO2 nanoparticles on germination, biochemical and morphoanatomical attributes of Cicer arietinum L. Energy Ecol Environ 2:277–288

    Article  Google Scholar 

  47. Zhou Q, Hu X (2017) Systemic stress and recovery patterns of rice roots in response to graphene oxide nanosheets. Environ Sci Technol 51:2022–2030

    CAS  Article  Google Scholar 

  48. Shen S, Liu Y, Wang F, Yao G, Xie L, Xu B (2019) Graphene oxide regulates root development and influences IAA concentration in rice. J Plant Growth Regul 38:241–248

    CAS  Article  Google Scholar 

  49. Li F, Sun C, Li X, Yu X, Luo C, Shen Y, Qu S (2018) The effect of graphene oxide on adventitious root formation and growth in apple. Plant Physiol Biochem 129:122–129

    CAS  Article  Google Scholar 

  50. Zhao G, Zhao Y, Lou W, Su J, Wei S, Yang X, Wang R, Guan R, Pu H, Shen W (2019) Nitrate reductase-dependent nitric oxide is crucial for multi-walled carbon nanotubes-induced plant tolerance against salinity. Nanoscale.

    CAS  Article  Google Scholar 

  51. Xuan W, Xu S, Li M, Han B, Zhang B, Zhang J, Lin Y, Huang J, Shen W, Cui J (2012) Nitric oxide is involved in hemin-induced cucumber adventitious rooting process. J Plant Physiol 169:1032–1039

    CAS  Article  Google Scholar 

  52. Sun A, Nie S, Xing D (2012) Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160:1081–1096

    CAS  Article  Google Scholar 

  53. Wang Y, Li L, Cui W, Xu S, Shen W, Wang R (2012) Hydrogen sulfide enhances alfalfa (Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351:107–119

    CAS  Article  Google Scholar 

  54. Li L, Wang Y, Shen W (2012) Roles of hydrogen sulfide and nitric oxide in the alleviation of cadmium-induced oxidative damage in alfalfa seedling roots. Biometals 25:617–631

    CAS  Article  Google Scholar 

  55. Xie Y, Mao Y, Zhang W, Lai D, Wang Q, Shen W (2014) Reactive oxygen species-dependent nitric oxide production contributes to hydrogen-promoted stomatal closure in Arabidopsis. Plant Physiol 165:759–773

    CAS  Article  Google Scholar 

  56. Zhang Y, Su J, Cheng D, Wang R, Mei Y, Hu H, Shen W, Zhang Y (2018) Nitric oxide contributes to methane-induced osmotic stress tolerance in mung bean. BMC Plant Biol 18:207

    CAS  Article  Google Scholar 

  57. Su J, Zhang Y, Nie Y, Cheng D, Wang R, Hu H, Chen J, Zhang J, Du Y, Shen W (2018) Hydrogen-induced osmotic tolerance is associated with nitric oxide-mediated proline accumulation and reestablishment of redox balance in alfalfa seedlings. Environ Exp Bot 147:249–260

    CAS  Article  Google Scholar 

  58. Li J, Zhu D, Wang R, Shen W, Guo Y, Ren Y, Shen W, Huang L (2015) β-Cyclodextrin-hemin complex-induced lateral root formation in tomato: involvement of nitric oxide and heme oxygenase 1. Plant Cell Rep 34:381–393

    CAS  Article  Google Scholar 

  59. Wang S, Uddin MI, Tanaka K, Yin L, Shi Z, Qi Y, Mano J, Matsui K, Shimomura N, Sakaki T, Deng X, Zhang S (2014) Maintenance of chloroplast structure and function by overexpression of the rice monogalactosyldiacylglycerol synthase gene leads to enhanced salt tolerance in tobacco. Plant Physio 165:1144–1155

    CAS  Article  Google Scholar 

  60. Han B, Yang Z, Xie Y, Nie L, Cui J, Shen W (2014) Arabidopsis HY1 confers cadmium tolerance by decreasing nitric oxide production and improving iron homeostasis. Mol Plant 7:388–403

    CAS  Article  Google Scholar 

  61. Miralles P, Johnson E, Church TL, Harris AT (2012) Multiwalled carbon nanotubes in alfalfa and wheat: toxicology and uptake. J R Soc Interface 9:3514–3527

    CAS  Article  Google Scholar 

  62. Ghosh M, Chakraborty A, Bandyopadhyay M, Mukherjee A (2011) Multi-walled carbon nanotubes (MWCNT): induction of DNA damage in plant and mammalian cells. J Hazard Mater 197:327–336

    CAS  Article  Google Scholar 

  63. Begum P, Ikhtiari R, Fugetsu B (2014) Potential impact of multi-walled carbon nanotubes exposure to the seedling stage of selected plant species. Nanomaterials 4:203–221

    Article  CAS  Google Scholar 

  64. Ghosh M, Bhadra S, Adegoke A, Bandyopadhyay M, Mukherjee A (2015) MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation. Mutat Res-Fund Mol M 774:49–58

    CAS  Article  Google Scholar 

  65. Wang F, Arai S, Endo M (2004) Metallization of multi-walled carbon nanotubes with copper by an electroless deposition process. Electrochem Commun 6:1042–1044

    CAS  Article  Google Scholar 

  66. Cañas JE, Long M, Nations S, Vadan R, Dai L, Luo MX, Ambikapathi R, Lee EH, Olszyk D (2008) Effects of functionalized and nonfunctionalized single-walled carbon nanotubes on root elongation of select crop species. Environ Toxicol Chem 27:1922–1931

    Article  Google Scholar 

  67. Stampoulis D, Sinha SK, White JC (2009) Assay-dependent phytotoxicity of nanoparticles to plants. Environ Sci Technol 43:9473–9479

    CAS  Article  Google Scholar 

  68. Del Castello F, Nejamkin A, Cassia R, Correa-Aragunde N, Fernandez B, Foresi N, Lombardo C, Ramirez L, Lamattina L (2019) The era of nitric oxide in plant biology: twenty years tying up loose ends. Nitric Oxide 85:17–27

    Article  CAS  Google Scholar 

  69. Lombardo MC, Lamattina L (2018) Abscisic acid and nitric oxide modulate cytoskeleton organization, root hair growth and ectopic hair formation in Arabidopsis. Nitric Oxide 80:89–97

    CAS  Article  Google Scholar 

  70. Beligni MV, Lamattina L (2000) Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210:215–221

    CAS  Article  Google Scholar 

  71. Yu M, Lamattina L, Spoel SH, Loake GJ (2014) Nitric oxide function in plant biology: a redox cue in deconvolution. New Phytol 202:1142–1156

    CAS  Article  Google Scholar 

  72. Stohr C, Stremlau S (2006) Formation and possible roles of nitric oxide in plant roots. J Exp Bot 57:463–470

    Article  CAS  Google Scholar 

  73. Besson-Bard A, Pugin A, Wendehenne D (2008) New insights into nitric oxide signaling in plants. Annu Rev Plant Biol 59:21–39

    CAS  Article  Google Scholar 

  74. Mur LAJ, Mandon J, Cristescu SM, Harren FJM, Prats E (2011) Methods of nitric oxide detection in plants: a commentary. Plant Sci 181:509–519

    CAS  Article  Google Scholar 

  75. Gas E, Flores-Pérez Ú, Sauret-Güeto S, Rodriguez-Concepcion M (2009) Hunting for plant nitric oxide synthase provides new evidence of a central role for plastids in nitric oxide metabolism. Plant Cell 21:18–23

    CAS  Article  Google Scholar 

  76. Gupta KJ, Fernie AR, Kaiser WM, van Dongen JT (2011) On the origins of nitric oxide. Trends Plant Sci 16:160–168

    CAS  Article  Google Scholar 

  77. Desikan R, Griffiths R, Hancock J, Neill S (2002) A new role for an old enzyme nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. Proc Natl Acad Sci U S A 99:16314–16318

    CAS  Article  Google Scholar 

Download references


Not Applicable.


This work was partially supported by the Natural Science Foundation of Jiangsu Province (Grant No: BK20181317).

Author information

Authors and Affiliations



ZYC and HZ contribute equally to this work. ZYC and HZ designed and performed this research. LSK, LNL, and RW carried out the related experiments and data analysis. HZ and WBS wrote the paper. All authors contributed to the general discussion and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Wenbiao Shen.

Ethics declarations

Competing Interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Figure S1.

Tg inhibits MWCNTs-induced NO accumulation. 3-day-old tomato seedlings were treated with distilled water and 5 mg/mL MWCNTs with or without 20 μM tungstate (Tg). The NO signal was analyzed by LSCM (A) and EPR (B) after treated for 24 h. Scale bar = 0.1 mm.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cao, Z., Zhou, H., Kong, L. et al. A Novel Mechanism Underlying Multi-walled Carbon Nanotube-Triggered Tomato Lateral Root Formation: the Involvement of Nitric Oxide. Nanoscale Res Lett 15, 49 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Multi-walled carbon nanotubes
  • Lateral root
  • Nitric oxide
  • Nitric reductase
  • Tomato