MWCNTs-Stimulated Root Hair Growth and the Distribution of MWCNTs
MWCNTs could affect plant growth and stress resistance. However, few studies focused on root hairs, and their conclusions are controversial [21, 22]. To provide the real picture regarding the function of MWCNTs in root hair development, two-day-old rapeseed seedlings were administrated with different concentrations of MWCNTs for 3 days. Compared with the control, all concentrations of MWCNTs (10, 50, 100, 200, and 500 mg/L) could differentially promote root hair development (Fig. 1a and b). Among these treatments, the response of 100 mg/L MWCNTs was maximal, and this concentration was subsequently used.
Our results matched with a previous observation, showing that MWCNTs (diameters ranging from 13 to 14 nm) could significantly promote root hair growth in 70, 80, and 90 mg/L in wheat . However, a controversial result was reported by García-Sánchez’s study , revealing that 25 mg/L COOH-MWCNTs (diameters ranging from 4 to 12 nm; and the modification increases the solubility of MWCNTs) could obviously inhibit root hair growth in Arabidopsis. We further compared the parameters of different MWCNTs used in previous reports and herein (diameters ranging from 6 to 12 nm), and found that the sizes of the three MWCNTs are almost the same. Therefore, it seems that different doses of MWCNTs and various plant species could be used to explain above mentioned difference.
To test our hypothesis, high concentrations of MWCNTs were applied either. As expected, the root hair development was seriously impaired by 2000 and 5000 mg/L MWCNTs, while no such significant decrease was found in the presence of 1000 mg/L MWCNTs (Supplementary Material, Fig. S1). Seven kinds of plant species, including rice, tomato, Chinese cabbage, wheat, radish, alfalfa, and Arabidopsis, were used subsequently. The results further revealed that appropriate concentrations of MWCNTs could differentially induce the growth of root hairs (Supplementary Material, Table. S3). However, lower and higher concentrations of MWCNTs normally had no such significant effect on root hairs or even inhibited their growth. Above results proved that the induction of root hairs induced by MWCNTs was a universal response.
The distribution of MWCNTs was further detected by using transmission electron microscopy. Consistent with the previous study , our results showed that the MWCNTs distributed in cytoplasm, intercellular space, and cell vacuole of rapeseed root tissues (Fig. 1c).
Ethylene was Involved in MWCNTs-Induced Root Hair Development
In most plants, ethylene signaling is indispensable in root hair development [25–29]. In order to assess whether ethylene participitates in MWCNTs-induced rapeseed root hair formation, we measured contents of endogenous ethylene in root tissues by using gas chromatography [27, 34]. In comparison with the control sample (Con), the time course analysis illustrated that the administration of MWCNTs for 18 h caused a progressive increase in the ethylene production, followed by peaking at 48 h and keeping a stable level until 72 h (Fig. 2a). Meanwhile, two critical enzymes for ethylene synthesis, 1-aminocyclopropane-1 -carboxylate (ACC) synthase and ACC oxidase, were also analyzed. As expected, similar tendencies were observed in above enzymatic activities (Fig. 2b and c). Ethylene synthesis in response to MWCNTs in rapeseed is also correlated to the biological response of MWCNTs control of root hair development (Fig. 1a and b).
To verify whether ethylene is involved in root hair development triggered by MWCNTs, two ethylene synthetic inhibitors cobalt chloride (CoCl2) [29, 30] and aminoethoxyvinylglycine (AVG) [30, 35] were used subsequently. Meanwhile, ACC, an ethylene synthesis precursor, was used as a positive control [28, 30]. As expected, 10 µM CoCl2 or 3 µM AVG alone, a concentration applied to individually decrease ACC oxidase [29, 30] and its synthetase activities [30, 35], not only impaired root hair growth (Fig. 2d and e), but also decreased ethylene content (Fig. 2f). Above results clearly confirmed the important function of endogenous ethylene in root hair development [25–28]. Further results revealed that MWCNTs control of root hair development was similar to the induction role of 3 µM ACC. By contrast, MWCNTs-induced root hair growth was inhibited by CoCl2 or AVG. Meanwhile, CoCl2 or AVG inhibition of MWCNTs-triggered ethylene was also observed, indicating a requirement for ethylene in MWCNTs control of root hair development. It is a new finding.
Similar results were discovered in the previous studies, showing that the removal of endogenous ethylene could impair root hair growth [25, 26], since ethylene is an essential signal for controlling root hair development, in either nutrient-adequate conditions [26, 28] or nutrient-starvation surroundings [27, 34].
NR-Dependent NO was Associated with MWCNTs-Induced Root Hair Development
Similar to ethylene [25–28], NO is another gaseous signal molecule in root hair development [31, 32]. In order to evaluate a possible interaction between NO and MWCNTs in root hair development, the endogenous NO signal in rapeseed roots were firstly detected by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), a membrane permeable probe used ubiquitously to monitor NO levels in plant tissues [33–35]. Figure 3a demonstrates that basal DAF-FM DA fluorescence was detected in chemical-free control samples, and that in the presence of MWCNTs, the fluorescence was significantly affected, showing the initial increase as early as 12 h, and reaching a peak at 48 h after treatment (Fig. 3a and Supplementary Material Fig. S2). These results clearly suggested that MWCNTs-induced NO was an early event.
In order to illustrate the primary source(s) of NO in our experimental conditions, the two main pathways of synthesizing NO in plants, NR and NOS [27, 31, 34, 36], were assessed. NR is the most intensively studied source of NO in plants.47However, although the plant NOS gene has not been successfully cloned, ample evidence that used the inhibitors of mammalian NOS, provided the evidence of L-arginine-dependent route in NO synthesis [45, 46]. As expected, the time course experiments found that in the presence of MWCNTs, the activities of NR showed the similar tendencies, as compared to the levels of NO production (Fig. 3b). However, no significant difference was observed in NOS-like enzyme activities of rapeseed seedlings with or without MWCNTs treatments (Fig. 3c). These results matched with the previous studies [13, 43], showing that endogenous NO is required for MWCNT-triggered plant tolerance against salinity stress, and further confirming that NR was the main enzymatic route for MWCNTs-induced NO, in either plant response against stress  or lateral root formation .
To investigate the mechanism underlying MWCNTs-elicited root hair development, NO-releasing compound SNP which was used as a positive control [34–36], NR inhibitor tungstate [40, 41], mammalian NOS inhibitor NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) [39, 40], and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxiden potassium salt (cPTIO) [40, 47], were used in the subsequent experiments. Among these, Old SNP, containing no NO, but nitrate, nitrite, and ferrocyanide, was further regarded as a negative control of SNP [40, 41].
When tungstate (in particularly), or L-NAME was applied alone, both root hair growth (Fig. 3d and e) and NO production (Fig. 3f) were simultaneously abolished, emphasizing the important function of endogenous NO in root hair development via NR, rather than NOS-like enzyme [48–50]. Meanwhile, we observed that an application of old SNP failed to alter root hair development as well as NO production. Subsequently, we showed that both MWCNTs- and SNP-promoted endogenous NO production and cPTIO, a NO scavenger. cPTIO alone also resulted in the decreased NO content and the significant reduction in root hair growth, further confirming the function of endogenous NO in root hair morphogenesis [31, 32]. In the presence of tungstate, MWCNT-induced NR activities were also significantly impaired (Fig. 3g), thus leading to a decreased NO production (Fig. 3f) and a reversed phenotype (Fig. 3d and e). Comparatively, since MWCNTs had no obvious effects on NOS-like activities (Fig. 3), and L-NAME could did not influence MWCNTs-induced NO (Fig. 3f) and root hair development (Fig. 3d and e). Those results thus suggested that NOS might not be the main source for NO production elicited by MWCNTs.
Combining above mentioned results, we further proved that MWCNTs-induced root hair development is dependent on NR-mediated NO synthesis.
Ethylene Acts Downstream of NO in MWCNTs-Induced Root Hair Growth
Previous Ample evidence revealed that NO and ethylene are key signaling molecules participating in various plant signal transduction processes. For example, both ethylene [25, 26] and NO [31–34] are individually suggested to induce root hair growth. However, the question of the relationship between ethylene and NO in MWCNTs-elicited root hair growth appears to be particularly interesting.
As shown in Figs. 2a and 3a, MWCNTs application stimulated the synthesis of NO and ethylene, and the initial inducible time points were 12 h and 18 h, indicating that NO might act upstream of ethylene in MWCNTs-triggered root hair development. To confirm this hypothesis, we further investigated the effects of two ethylene synthetic inhibitors CoCl2 and AVG on the NO content; and NO scavenger cPTIO, its synthetic inhibitors tungstate and L-NAME, on the ethylene content.
Interestingly, administration with CoCl2 and AVG, two ethylene synthetic inhibitors, did not alter NO synthesis, including its content, NR and NOS activities, under the conditions in the presence and absence of MWCNTs (Fig. 4a-d). Unlike the responses of NOS inhibitor L-NAME, both NO scavenger cPTIO and NR inhibitor tungstate could obviously block ethylene synthesis triggered by MWCNTs (Fig. 4e-g). However, when exogenously applied alone, including cPTIO, tungstate, and even L-NAME, could decrease ethylene content, and inhibit ACC synthase and oxidase activities, reflecting the fact that both NR and NOS-like enzyme are responsible for NO synthesis in rapeseed seedlings under the normal growth conditions. Similar findings were reported in other plant species, including Arabidopsis , red kidney bean , and barley .
So, by combining data from Figs. 2–4, we concluded that some linearity may exist in NO and ethylene signalling downstream of MWCNTs. Interestingly, a similar relationship of NO and thereafter ethylene signalling was observed in the adaptive response of phosphorus-deficient rice . And, the interaction relationship between NO and ethylene was found in a magnesium-deficiency condition . Therefore, it could be suggested that plant response to an external stimulus is controlled by a complex array of signalling mechanisms, and plants respond differently to various environmental stimuli with the same signals, in a linear fashion or a cross-talk manner.
MWCNTs-Modulated Transcripts Related to Root Hair Development were Dependent on NO and Ethylene Synthesis
It is well documented that the TRANSPARENT TESTA GLABRA (TTG), GLABRA2 (GL2), and GLABRA3 (GL3) could accelerate the development of epidermal cells to nonhair cells, thus resulting in the inhibition in the initiation of root hair development [51, 52]. The Rho-related GTPase from plants (ROP) is closely associated with root hair initiation and tip growth [53, 54]. Correspondingly, CAPRICE (CPC) and TRIPTYCHON (TRY) genes are known to trigger root hair formation [51, 52]. Auxin signals AUXINRESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) participate in ethylene- and NO-elicited root hair formation via regulating corresponding marker genes related to root hair development [34, 55, 56].
Accordingly, above molecular marker genes related to root hair growth were detected by qPCR. Like the responses of ACC and SNP, the expression of BnAUX1, BnPIN1, BnCPC, and BnTRY were up-regulated in MWCNTs-induced rapeseed root hair growth, while the expression of BnTTG and BnGL2 were down-regulated (Fig. 5). Above responses could differentially blocked or impaired by the addition of CoCl2, AVG, cPTIO, tungstate, indicating that both ethylene and NO are required for the modulation of these molecular marker genes in MWCNTs control of root hair development. Similarly, previous results also revealed that the genes related to root hair growth were regulated in magnesium deficiency-promoted root hair growth via ethylene and NO signals . Above results indicated that MWCNTs-induced root hair growth is closely associated with the adjustment of the marker gene expression.
Genetic Evidence Revealed that Ethylene and NR-Dependent NO were Associated with MWCNTs-Induced Root Hair Development
Pharmacological experiments may not fully reflect the true roles of endogenous ethylene and NO signals in root hair development, and may have side effects . So, the genetic mutants of Arabidopsis were subsequently used. Arabidopsis is a model plant which belongs to the cruciferous family, and have high homology of rapeseed . The mutants used in the experiment were NO related mutant nia1/2 (exhibited impaired nitrate reductase activity) and noa1 (encoding NO-associated protein 1; with indirectly reduced NO level in vivo), and ethylene related mutant ein2-5 (ethylene-insensitive mutant) and ein3-1 (ethylene-insensitive mutant).
Previous reports discovered that nia1/2, noa1, ein2-5, and ein3-1 showed poor root hair growth [27, 34]. Similar results were confirmed in our study, showing lower root hair density in these four mutants, and shorter root hairs in ein2-5 and ein3-1 (Fig. 6a-c), indicating that endogenous ethylene and NO function in plant root hair growth.
For nia1/2 and noa1 mutants, under the control condition, the obvious reduction in NO and ethylene contents (Fig. 6d and f), matched with the phenotypes of the root hairs, compared to the wild-type (WT) plants (Fig. 6a-c). Biochemical analysis further showed that unlike the data in nia1/2 mutant, although NR activity was not altered in noa1 mutant, the similar and significant reduction in NO content was observed. While, no such significant decreases in the NO and ethylene signals were observed in ein2-5 and ein3-1 mutants. These could be explained by the fact, that both ein2-5 and ein3-1 are ethylene-receptor-related mutants that have little effect on endogenous ethylene [58–62]. Since the decreased ethylene contents were also observed in nia1/2 and noa1 mutants (Fig. 6e and g), it further provides genetic evidence for the function of endogenous ethylene as a downstream signal of NO governing root hair growth.
Subsequent experiments discovered that the application of MWCNTs could induce root hair growth in wild type (WT) and noa1 mutant, reflecting the important function of NR in above response. Consistently, a significant reduction in root hair development was found in nia1/2 mutant, when challenged with MWCNTs (Fig. 6a-c). Similar phenomenon was observed in ein2-5 and ein3-1 mutants, also indicating the role of ethylene signalling.
Related ethylene and NO synthesis were also examined in above materials. As anticipated, changes in ethylene and NO contents matched with phenotypes, showing higher ethylene and NO contents in MWCNTs-treated WT, noa1, ein2-5, and ein3-1 mutants, which different from the impaired ethylene and NO contents in nia1/2 mutant (Fig. 6d and f). Thus, combined with the phenomenon in root hair development (Fig. 6a-c), above genetic evidence strongly suggested that NR-dependent NO and ethylene were required for MWCNTs-induced Arabidopsis root hair development, consistent with our previous findings in rapeseed by pharmacological, biochemical, and molecular approaches (Figs. 1–5).
The cross talk between ethylene and NO was further investigated. As shown in Fig. S3, by using tungstate, an inhibitor of NR, and AVG, an inhibitor of ACC synthetase, we observed that the removal of the major known sources of NO or ethylene severely impaired MWCNTs-induced ethylene production and thereafter root hair formation. These further indicated a requirement for ethylene in above MWCNTs responses. Moreover, since AVG did not affect NO production, we further confirm that ethylene acts downstream of NO signalling in MWCNTs governing root hair morphogenesis.
Auxin, a well-known phytohormone, has an important function in controlling root hair growth [31, 34]. Previous genetic evidence discovered that auxin might function downstream of ethylene and NO signaling to promote Arabidopsis root hair formation under magnesium deficiency conditions . It is well documented that auxin transport is mediated by AUXINRESISTANT1 (AUX1)/LAX influx carriers and the PIN-FORMED (PIN) efflux carrier family [53, 54]. For example, it was documented that AUX1 could increases efficiency of auxin uptake, thus resulting in the efficient transport of auxin and its accumulation within plant tissues. Here, we further evaluated the roles of auxin signaling in MWCNTs response, and found that YFP and GFP fluorescence in the roots of AUX1::AUX1-YFP and PIN1::PIN1-GFP transgenic plants were increased by MWCNTs, both of which could be differentially impaired in the presence of either tungstate or AVG (Supplementary Material, Fig. 7a and b). The changes of AtAUX1 and AtPIN1 transcripts were also confirmed by using qPCR in WT, nia1/2, and ein2-5 under normal or MWCNTs-treated condition, showing the decreasing tendencies in two mutants, especially upon MWCNTs (Supplementary Material, Fig. 7c and d). These results also indicated that auxin might be controlled by ethylene and NO in MWCNTs control of Arabidopsis root hair development, which should be further assessed in the near future.
Furthermore, representative genes, including AtCPC, AtTRY, AtROP2, AtTTG1, AtGL2, and AtGL3, were analyzed [52–54]. Compared to the WT, in either the control conditions or in the presence of MWCNTs (in particularly), the down-regulated AtCPC (except in the control conditions), AtTRY and AtROP2, and the up-regulated AtTTG1, AtGL2, and AtGL3 in both nia1/2 and ein2-5 mutants were observed (Supplementary Material, Fig. 8), all of which could be used to explain the defected root growth phenomena (Fig. 6a-c), since AtTRY and AtROP2 belong to root hair growth promoting factors, and AtTTG1, AtGL2, and AtGL3 are negative regulators [51, 52]. This genetic and molecular evidence also pointed out that the molecular maker genes associated with root hair growth could be modulated by MWCNTs via NO-ethylene pathway.
Combining above results in rapeseed and Arabidopsis, we proposed that both ethylene and NO were required for MWCNTs-induced root hair morphogenesis, and ethylene might act downstream of NO in the regulatory cascade. The involvement of auxin signalling was also suggested. Related model was summarized in Fig. 9.