Carbon Nanoparticles Enhance Potassium Uptake via Upregulating Potassium Channel Expression and Imitating Biological Ion Channels in BY-2 Cells

doi:10.21203/rs.2.18223/v1

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Carbon Nanoparticles Enhance Potassium Uptake via Upregulating Potassium Channel Expression and Imitating Biological Ion Channels in BY-2 Cells

https://doi.org/10.21203/rs.2.18223/v1

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published 28 Jan, 2020

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Background: Carbon nanoparticles (CNPs) have been reported to boost plant growth, while CNPs enhancing potassium uptake for plant growth have not been reported so far.

Results: In this study, the function of CNPs promoting potassium uptake in BY-2 cells was established and the potassium accumulation in cells has a significant correlation relationship with BY-2 cell fresh biomass. The K + accumulation in cells increased with the increasing concentration of CNPs. The K + influx reached high level after treatment with CNPs and exhibited significantly higher than the control group and the negative group treated with K + channels blocker, tetraethylammonium chloride (TEA + ). The K + accumulation was not reduced in the presence of CNPs inhibitors. In presence of the potassium channel blocker TEA + and the CNPs inhibitors, NKT1 expression was downregulated. The CNPs were found to preferentially transport K + than other cations determined by rectification of ion current assay (RIC) in a conical nanocapillary.

Conclusions: These results indicated that CNPs upregulated potassium gene expression to enhance K + accumulation in BY-2 cells. Moreover, it was speculated that the CNPs simulated protein ion channels via bulk of carboxyl for K + permeating. These findings will provide support for improving plant growth by carbon nanoparticles.

Nanoscience

carbon nanoparticles

potassium uptake

gene expression

ion channels

Potassium is an essential macronutrient for all plants growth[1–3]. It is abundant in plant cells and is required for multiple functions, such as maintenance of osmotic pressure, activation of numerous enzymes, and enhancement of photosynthesis. It is well established that K⁺ uptake is characterized by biphasic uptake kinetics, a high-affinity transport system (mechanism 1, i.e., transporters) and a low-affinity transport system (mechanism 2, i.e., K⁺ channel) [4]. At low external concentrations of K⁺ (≤ 0.2 mM), the mechanism 1 works against an electrochemical gradient, by consumption of energy provided by ATP. The mechanism 2 operates mostly via the activity of K⁺ channels at high external concentrations without energy (≥ 0.2 mM).

For the past few years, researchers had increasing interest in promoting the crop production by applying with carbon nanomaterials[5–6]. Carbon nanomaterials were proved useful in seed germination[7], root growth[8] and photosynthesis[8–9]. Carbon nanotubes can stimulate growth, gene and protein expression of aquaporin in tobacco cells[10]. Low dose multi-walled carbon nanotubes (MWCNTs) were proved to be beneficial in maize, improving water absorption, plant biomass and the content of the essential Ca, Fe nutrients uptake[11]. Potassium plays an important role in plant growth. In previous reports, researchers mainly focus on changes of plant biomass, rarely on the relationship between nanomaterials and potassium accumulation in plants. Therefore, uncovering the relationship between nanomaterials and potassium accumulation in plants will better clarify the mechanism of carbon nanomaterials for promoting plant growth.

Whether carbon nanomaterials can facilitate K⁺ uptake in biological system, researchers hold quite the opposite opinions. Some researchers believe that carbon nanomaterials suppress potassium channel activities and block K⁺ uptake in mammalian cells[12–15]. C₆₀ neat fullerenes was found to hinder the function of K⁺ channels by binding with K⁺ channel proteins via computer simulations methods[16]. MWCNTs inhibited potassium channel activities in PC12 cells and CHO cells via current decrease in the whole-cell electrophysiological recording[17]. Others insist that carbon nanomaterials are able to enhance K⁺ uptake in biological system, although there are very rare reports. Graphene oxide (GO) was found to be internalized into primary astrocytes and upregulated inward-rectifying K⁺ channels and Na⁺-dependent glutamate uptake[18]. Single-Walled Carbon Nanotubes (SWCNTs) mimic potassium channels for K⁺ uptake by biomimetic devices[19]. The effect of carbon nanomaterials on K⁺ uptake was conflicting related to diverse and stable nanostructures. Carbon nanotubes were formed a cylindrical channel with a diameter of 1 nm by a graphene sheet, and the carbonyl on the entrance of channel preferentially permitted K⁺ pass through. While C60 fullerene with stable spherical structure, has an average diameter of 0.72 nm, which may fill in the ion channels and block K⁺ entering cells[12]. Whereas, there are little reports about carbon nanomaterials enhancing K⁺ uptake in plant cells.

BY-2 cells are used for K⁺ uptake research, due to growing rapidly and being synchronized, especially requirement for amount of K⁺ for growth.[20–21] In addition, Shaker-like K⁺ channel genes in BY-2 cells were confirmed and named NKT1, NtKC1 and NTORK1. The inward rectifying NKT1 channels were involved in cell division through K⁺ uptake into cells, and outward rectifying K⁺ channel NTORK1 activity was concerned with cell cycle in Xenopus oocytes[20–21]. The transporter gene NtHAK1 was involved in K⁺ uptake under low-K⁺ conditions.

In this study, we employed BY-2 cell for nanobiotechnology research to elucidate the mechanisms of CNPs function on promoting K⁺ accumulated content and these results revealed that the mechanism of CNPs enhenced BY-2 cells growth for further. The proposed molecular structure of CNPs (Fig. 1) was drawn according to previus characteration[22–23]. BY-2 cells were incubated with different concentrations of CNPs. We hypothesized that the genes involved in K⁺ uptake were modulated by CNPs to increase the activities of K⁺ influx channels. We measured changes of gene expression and K⁺ channel activity in BY-2 cells. The ion selectivity of CNPs was also evaluated by filled in the conical nanocapillary. The effect of CNPs on plant cell performance was assessed, including changes in intracellular amino acid content and pH value of extracellular matrix. Here, we demonstrated that the concerned K⁺ uptake genes were upregulating by CNPs. In addition, we deduced that CNPs embed on the membrane and acted as biological ion channels for K⁺ permeating. These findings also provide explaination for carbon based nanomaterias promoting plant growth for further.

Material

Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) suspension cells were cultured with Murashige and Skoog (MS) liquid medium supplemented with sucrose (30 g/L) and 2,4-dichlorophenoxyacetic (2,4-D, 1 mg/L). The suspension cells were sub-cultured at 4-day intervals by transferring 20 mL of suspension into 40 mL of fresh medium, agitating on a rotary shaker (133 rpm, 27.5℃) in the dark. CNPs were prepared as described in Chen et al[22].

Determination of cell mass and K ⁺ content in cells

Suspension cells were filtered from the MS liquid medium by cell strainer (40 µm, Falcon). Then the cells were washed three times to remove the extracellular K⁺ with 3% sucrose solution. After that cells were centrifuged at 200 × g for 3 min and the fresh mass of cells was recorded. Then the BY-2 cells were resuspended in ultrapure water. For K⁺ extraction, cells were broken by ultrasonication (JY92-2D, Scientz, China). The supernatant was collected, and K⁺ concentrations were determined using inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7500a, USA).

TGA-FTIR analysis of CNPs

The pyrolysis of CNPs and CNPs treated with ethanol were determined by using thermogravimetric analysis (TGA) coupled to a Fourier transform infrared spectrum (FTIR) technique. The powder samples of 8 mg were placed in small PtHT crucibles. The TGA pyrolysis program operated with a nitrogen atmosphere (100 ml/min). The samples were heated uo to 100 °C and equilibrated for 20 min to remove the H₂O and air atmosphere.The heating rate was set to 15 °C/min from 100 °C up to a final temperature of 800 °C. The generated gas phase volatiles were transfered via a heated line (230 °C) interfaced to the FTIR.

K ⁺ uptake related genes expression

Total RNA of BY-2 cell was extracted and purified using RNeasy plant mini kit (Sangon Biotech, China). Complementary DNA was synthesized using M-MLV reverse transcriptase (Takara) with oligo (dT) primers. These K⁺ uptake related gene primers were synthetic according to the National Center for Biotechnology Information database (NCBI ), including the inward-rectifying K⁺ channel NKT1 (GenBank: AB196790) and NtKC1 (GenBank: AB196791), outward-rectifying K⁺ channel NTORK1 (GenBank: AB196792), plasma membrane H⁺-ATPase NHA1 (GenBank: AY383599) and K⁺ transporter NtHAK1 (GenBank: DQ841950). Real-time quantitative PCR was performed on a CFX96 real-time system (Bio-RAD, USA) with three steps using LightCycler® 480 SYBR Green I Master (Roche) according to the manufacturer’s instructions. The relative transcript level of genes was quantified by 2^−∆∆CT method[24]. The control gene (Actin) was used as normalization for the test gene transcript.

Non-invasive Micro-test Technology (NMT) for potassium ion flux analysis

Net fluxes of K⁺ were measured using NMT system (Younger USA LLC, Amherst, MA01002, USA) [25–26]. BY-2 cells were incubated with bath solution (46.88 mM KCl, 0.1 mM CaCl₂, 0.3 mM Mes, pH 5.9 unbuffered) in petri dish. The work microelectrode was positioned in close proximity to a selected BY-2 cell and left to equilibrate for a few minutes. The micropipettes were filled with a length of 180 µm of selective liquid ion-exchange cocktails (K⁺ LIX, XY-SJ-K, Younger, USA). The microelectrode is stepped from one position to close to the selected cell in a predefined sampling routine. Pre-pulled and silanized microsensor (Φ1.5 ± 0.5 µm, XY-CGQ-02, Younger USA) were filled with a backfilling solution (100 mM KCl). An Ag/AgCl wire microsensor holder YG003-Y11 (Younger, USA), was used as the reference microsensor. The exported raw data of micro-volt differences (△µV) were then imported and converted into net K⁺ fluxes by using the JCal V3.3 (a free MS Excel spreadsheet, youngerusa.com or xbi.org)

Ion Current Measurements

Glass capillaries were pulled by Sutter 97 to prepared conical nanocapillaries. Two Ag/AgCl electrodes were used to record a current-voltage curve via a patch-clamp amplifier[27]. One electrode is placed in the reservoir, facing the nanocapillary. The other one was inserted into the capillary. The conical nanocapillaries were filled with CNPs solution with pure water as a control and GO as a positive control. Transport properties of nanocapillary were measured in KCl, NaCl, NH₄Cl and CaCl₂ solution at the concentration of 100 mM.

Free amino acids analysis

The cell ultrasonication lysis supernatant was also collected for amino acids analysis using amino acid analyzer (Biochrom 30+, Biochrom Ltd, UK). The Li buffer was added to supernatant for adjustment the pH value. Amino Acid Standard (Sigma) was used to quantify the concentration of amino acids in lysis supernatant.

Measurement of extracellular matrix pH value

Cells were incubated variation concentration of CNPs for 12 h. Extracellular matrix were separated from cells by cell strainer and collected for pH value determination.

K ⁺ uptake in BY-2 cells

The properties of K⁺ uptake in BY-2 cells were investigated. As shown in Fig. 2, intracellular accumulated K⁺ content increased with the addition of KCl concentration and reached a relative maximum value at a concentration of 93.75 mM/L. Then intracellular accumulated K⁺ content was no longer increasing. Intracellular K⁺ accumulation was also found to be related to the incubation time of KCl. The intracellular K⁺ accumulation as well as increased with the prolonged incubation time, and was significantly different compared with control group when concentration of K⁺ was lower than 93.75 mM/L. The intracellular K⁺ accumulation was increased with the addition of variable concentration of K⁺, which was similar with barley roots K⁺ uptake from 0.2–50 mM/L of KCl treatment[4]. In previous publications, K⁺ accumulation in plant cells (BY-2 cells) reached a threshold and then initiated the next cell cycle. Division cycle of BY-2 cells took approximate 12 h[21]. This explained that the higher intracellular K⁺ accumulation was due to the multiples incubation time of division cycle.

In Fig. 3A, addition of different concentration of CNPs and a fixed K⁺ concentration of 46.88 mM/L in BY-2 cells culture medium, K⁺ accumulation in cells was enhanced with the addition of CNPs. When concentration of CNPs reached 62.50 µg/mL, intracellular K⁺ accumulation was almost 40 µΜ/g cells and remarkable difference with rising 21.12% compared to control group (with 46.88 mM/L KCl and without CNPs). There was no obvious difference in K⁺ accumulation in cells between the incubation time of 12 h and 24 h. The cell fresh biomass increased with addition of CNPs (Fig. 3B). As shown in Fig. 3C, cell fresh biomass exhibited correlation relationship with K⁺ accumulation in cells (12 h). As shown in Fig. S1A, CNPs did not affect the intracellular K⁺ accumulation under the condition of no addition of K⁺. The intracellular K⁺ accumulation displayed significantly different when the concentration of CNPs reached 125.00 µg/mL incubated with 20 mM/L of KCl for 12 h and 24 h (Fig. S1B). These results indicated that CNPs exerted influence in the presence of a certain concentration of K⁺.

The CNPs treatment with ethanol did not promote K⁺ accumulated in BY-2 cells in Figure S2A (ethanol was removed from CNPs). When ethanol was added to the CNPs solution with a proportion of 2:1 (v:v), the CNPs aggregated to form larger particles and precipitated in the mixed solution. The CNPs were resuspended in the pure water after removing ethanol, and the dispersibility became worse than the original CNPs. In the IR spectra of CNPs, the broad peak located from 3200 − 2500 cm^− 1 was assigned to dimer carboxyl. When CNPs were treated with ethanol, the carboxyl characteristic peaks appeared to be wide, from 3200 to 1900 cm^− 1. Besides, the intensity of C = O and the C = C in aromatic ring (the common feature of graphitic sp² hybridization carbons) located at 1680 and 1650 − 1450 cm^− 1 was changed compared with that of CNPs (Fig. S2B). The differences between CNPs and CNPs treated with ethanol also were assessed by a thermogravimetric analysis (TGA) coupled to a Fourier transform infrared spectrum (FTIR). TGA and DTG thermograms of the pyrolysis displayed obvious difference in CNPs and CNPs treated with ethanol in Figure S2C and S2D. There is a higher weight loss rate and more pyrolysis stages in CNPs than that in CNPs treated with ethanol. In the FTIR spectra (Fig. S2E and S2F), the formation of CO₂ at different stages originates from the fracture and recombination of carbonyl and carboxyl group (corresponding to different distribution, as shown in Fig. 1), while the generated CO is derived from the rupture of carbonyl and ether bond.[28] The carbonyl and carboxyl group on CNPs was disrupted by ethanol from the TGA-FTIR analysis. Therefore, it was speculated that the carboxyl groups were responsible for facilitating K⁺ uptake in BY-2 cells.

K ⁺ uptake related genes expressed in BY-2 cells

Inward-rectifier NKT1, NtKC1 and outward-rectifier NTORK1 was involved in K⁺ uptake identified in BY-2 cells. NKT1 and NTORK1 gene expression was upregulated in a linear manner with the increasing CNPs concentration, while NtKC1 gene expression was downregulated (Fig. 4A, 4B and 4C). The plasma membrane H⁺-ATPase NHA1 and K⁺ transporter NtHAK1 was also upregulated (Fig. 4D and 4E). Results of quantitative PCR demonstrated that NKT1, NHA1 and NtHAK1 relative transcripts exhibited significant difference and were at least 2-fold higher compared with control and other treated groups at a concentration of 62.50 and 125.00 µg/mL. Gene expression analysis results were basically in agreement with the results of K⁺ content accumulation in cells. These results indicated that NKT1 channels, NHA1 and NtHAK1 play an important role in K⁺ uptake enhanced by CNPs. Potassium channel NKT1 exert high-capacity K⁺ uptake in BY-2 cells, which represents a low-affinity with the external K⁺ concentration greater than 0.2 mM. When external K⁺ concentration was in the micromolar range from 0.002-0.2 mM, high-affinity systems worked, such as K⁺ transporter NtHAK1. It has been reported that K⁺ transporter seemed to be less important for K⁺ uptake in BY-2 cells. The plasma membrane H⁺-ATPase was found to maintain a proton gradient in plants[29] and fungi[30]. Overexpression of H⁺-ATPase AHA2 promotes hyperpolarization of the plasma membrane of guard cell in response to blue light, and increasingly induces K⁺ uptake into guard cells in A. thaliana transgenic plants[31].

Effect of CNPs on K ⁺ flux in BY-2 cells

The net K⁺ flux response to CNPs exposure was measured using the NMT technology to further understand the mechanism of promoting potassium accumulation in BY-2 cells. As shown in Fig. 5A, the K⁺ influx reached high level after treatment with variation concentration of CNPs for 12 h exposure, and exhibited significantly higher than the control group and the negative group treated with K⁺ channels blockers of TEA⁺ (p༜0.05, n = 6). The average K⁺ influxes ranged from the maximum value 3797.50 ± 942.46 to the minimum value 1372.95 ± 196.11 pmol·cm²·s^− 1 and the highest value of K⁺ influx treated with CNPs almost was a threefold and twofold increase compared with the control and the negative control group, respectively (Fig. 5C). There was no obvious significance between the negative control and control group. The K⁺ influx increased by CNPs application in NMT assay probably related with transmembrane ion channels. To verify whether the observed K⁺ caused by the activity of K⁺ channels, BY-2 was pretreated with 10 mM tetraethylammonium chloride (TEA⁺), a well known inhibitor of K⁺ channels. After 12 h of TEA⁺ treatment, K⁺ influx was inhibited in the presence of CNPs. Based on these data, it could be concluded that main part of the net K⁺ influx originated from the activity of plasma membrane K⁺ channels NKT1 induced by CNPs.

Effect of K ⁺ channel blockers and CNPs blocker on K⁺ uptake

Treatment with TEA⁺ (10 mM) exhibited a significant decrease in K⁺ accumulation in cells compared with CNPs treated group, while there was no significant difference with control groups (Fig. 6A). The results indicated that K⁺ uptake was inhibited by TEA⁺ to some extent. No obvious effect of K⁺ accumulation was found by addition of genistein or methyl-β-cyclodextrin. Gene expression analysis results showed that K⁺ channel blockers and CNPs blockers downregulated gene transcripts and was not in accord with results of K⁺ accumulation in cells (Fig. 6B-6F). Genistein and methyl-β-cyclodextrin were proved to inhibit CNPs entering BY-2 cells, which demonstrated that another pathway of CNPs promoting K⁺ uptake was not via entering BY-2 cells.

Rectification of Ion Current in Nanopores

Conical nanopores with a dissymmetry of charge or shape are also particularly fascinating for their non-linear rectification property, similar to ion channel on the cell membrane. I-V curves were shown for the ionic selectivity characteristic of the conical nanopores in the absence or in the presence of CNPs, GO as a positive control (Fig. 7A-7C). The Rectification of Ion Current (RIC) ratio calculated from the non-linear I-V curve of K⁺ is up to 4.7 higher than other cation in the presence of CNPs (Fig. 7E). NH₄⁺ exhibited high selectivity when conical nanopore was filled with GO (Fig. 7F). There was no selectivity among the monovalent cation and divalent cation in conical nanopores infused with pure water (Fig. 6D). Substantial difference of the ion selectivity may attribute to the chemical characteristics induced by CNPs or GO, neglecting the conical nanopore shapes in these cases. Therefore, it was speculated that carboxyl groups may imitate the potassium channels for K⁺ uptake by loading on the membrane of BY-2 cells.

The structure of ion channels provides guidance for biomimetic nanopores designing. Charged carboxyl of amino acid side chains plays a vital role in the selectivity properties of biological ion channels. Carbon nanotubes have been used for biomimetic nanopores research due to negative charged carboxyl. Single-walled carbon nanotubes (SWCNTs) were used for the ion conductance and found to be mimics of ion channels for K⁺ permeating[19]. These negatively charged carboxylates at the entrance of the SWCNTs exerted enormous impact on the ion transport, especially on K⁺ permeability. Moreover, SWCNTs have been designed as biomimetic nanopores for ion selectivity based on voltage-gated ion channels[32]. Though there are carboxyl groups on the GO, GO did not perform high K⁺ selectivity due to the distribution of carboxyl groups on GO. Single graphene nanopore loaded on PET membrane (G/PET nanopore) with high ionic flux and high ion selectivity was confirmed in the G/PET nanopore system[33].

Effect of CNPs on Amino acid content in BY-2 cells

Amino acids metabolites were as an indicator for CNPs improvement of K⁺ uptake in BY-2 cells. The metabolites of 25 kinds of amino acid from BY-2 cells were identified and were analyzed to determine the effect of CNPs exposure, as shown in Table 1. Amino acid content of the CNPs treated groups was significantly different from that of the control group, which was upregulated or downregulated in the four exposure groups compared with the control groups. 12 kinds of amino acid content were increased more than two-fold with the increasing concentration of CNPs, and decreased at the concentration of 125.00 µg/mL, including L-proline, L-arginine and glycine, etc. However, the content of L-tyrosine was reduced with the increasing concentration of CNPs. The content amino acids were changed under BY-2 cells response to CNPs stress condition.

Proline adjusted plant to stress response[34]. It had been reported that many plants accumulated proline under stress conditions (drought, low and high temperatures, high salinity) [35–36]. Proline not only is an osmotic regulator in plant cytoplasm, but also plays an important role in stabilizing the structure of macromolecules, reducing cell acidity, removing ammonia toxicity, and regulating cell redox[37]. Arginine plays an important role in the synthesis of polyamines that acts as the signaling molecule and improves stress resistance during defensive reaction in plants. Its content increased more than two times compared with control group, indicating its involvement in BY-2 cells metabolism under CNPs exposure condition. Increasement of amino acid content is vital for plants during stress condition, and our results were in agreement with the previous statement. Glycine promotes the uptake of phosphorus and potassium in plants, enhances plant stress resistance and improves the activity of enzymes. The variation trend of glycine was consistent with that of K⁺ accumulation in cells promoted by CNPs.

Effect of CNPs on external medium pH

The high concentration of CNPs (4 mg/mL) had a low pH value of 2.00. While addition of CNPs caused no obvious change in pH value of culture medium with higher concentration of CNPs incubated for 12 h (P༜0.05), as shown in Fig. S3. The BY-2 cells may produce secondary metabolites to resist the pH value decrease. The suitable pH value in BY-2 cells culture medium is crucial for microbial growth, metabolism, and biosynthesis of secondary metabolites[38].

The intracellular K⁺ accumulation was determined by ICP-MS and found to be increased significantly compared with control groups with CNPs concentration of 61.25 µg/mL. Subsequently, the K⁺ uptake related genes, including potassium channels and transporter genes, were upregulating in comparison with control groups analyzed by RT-PCR especially the inward-rectifying potassium channels gene NKT1. The CNPs increased K⁺ accumulation in BY-2 cells via upregulating the K⁺ uptake related genes. The trend of K⁺ increased content was basically in agreement with the genes upregulating with the increasing concentration of CNPs. K⁺ entering cells via potassium channels belongs to passive transport, independent of energy. The way of potassium transporters are ATP-dependent. In previous study, CNPs were found to enter BY-2 cells and aggregated around the cell nucleus, which accounted for genes expression. In addition, CNPs were also confirmed no effect on ATP production[23]. TEA⁺ could significant inhibit K⁺ uptake in the presence of CNPs, which demonstrated that potassium channels play an important role in K⁺ uptake. Although NtHAK1 gene encoding K⁺ transporter was upregulated, whose transport activity is dependent on the K⁺ electrochemical gradient across plasma membrane, the NtHAK1 active transporter contribution to K⁺ accumulated in cells was not significant compared to NKT1 channels. In the presence of potassium channels blocker TEA⁺, the K⁺ accumulation significantly decreased with the CNPs treated group, but that was no obvious difference with the control group, which indicated the contribution of NtHAK1 gene seemed to be less important. These results elaborated the mechanism of CNPs promoting K⁺ accumulation in cells. H⁺-ATPase NHA1 pumps H⁺ from the cell cytosol to the extracellular, building up the electrochemical gradient to form the potential between the inside and outside of plasma membrane[39], which was proved less important than NKT1 for K⁺ uptake.

CNPs exhibited particulate morphology with an average size of 30 nm. There are amount of groups of COOH and OH present on the surface of CNPs, and CNPs showed the common feature of graphitic sp² hybridization carbons[22]. The CNPs with disturbing carbonyl by ethanol were proved no impact on potassium uptake. Therefore, it was speculated that the carbonyl was crucial for K⁺ uptake. It has been reported that negatively charged nanotubes have a significant impact on both seed germination and plant growth, especially carbonyl on nanotubes surfaces[40]. According to the published reports, potassium ion channels usually are composed of four subunits of protein, which are symmetric to form a K⁺ selectivity filter for single potassium ion to pass through[41]. The K⁺ selectivity filter consists of amino acid sequence TVGY, which are distributed nearly evenly over the four binding sites. Each ion-binding site is surrounded by eight oxygen atoms contributed by the backbone carbonyl groups of the amino acid in TVGY sequence and the side-chain of the threonine residue[42]. The carbonyl oxygen atoms from amino acids point to the pore with a diameter of 0.3 nm, which allows K⁺ to pass through, but inhibits Na⁺, Ca²⁺ and NH₄⁺ or other ions going through. It has been confirmed that there was large amount of carbonyl on the surface of CNPs. Although the CNPs were inhibited to enter BY-2 cells by methyl-β-cyclodextrin and genistein gathering on the outside of membrane, K⁺ accumulated content was not decreased compared with the CNPs treated group. In addition, the potassium channel gene NKT1 downregulated in the presence of methyl-β-cyclodextrin, inhibiting CNPs entering BY-2 cells. Moreover, the CNPs loaded in the nanopore exhibited K⁺ selectivity compared with other cations using the RIC assay. Therefore, the carbonyl may presumably form nanopores similar to potassium channels on the membrane to permit K⁺ penetrating into cells. These results indicated that the CNPs may interact with membrane by simulating biological ion channels for K⁺ uptake, which was another mechanism for K⁺ uptake.

The potential benefits of employing metabolomics have recently been recognized to assess the toxicology of nanomaterials applied in the biological system. The effect of CNPs exposure on the amino acid metabolic profile was determined, and 25 kinds of amino acid were analyzed. The amino acid metabolites were upregulated or downregulated in the four exposure groups compared with the control group. CNPs reduced the levels of L-Tyrosine and boosted content of some amino acid, such as L-aspartic acid, L-serine, and L-proline with the increasing concentration of CNPs in cells. The proline content was influenced by the potassium levels to a large extent. Generally, as potassium levels increased, the production of proline showed to rise[43]. It has been reported that the downregulation of amino acid metabolism is associated with the enhancement of oxidative stress.[6] With upregulating of some amino acid, especially L-proline, the oxidative stress appeared to be alleviated in spite of presence of CNPs. These results were consistent with previous publication, the production of ROS was at lower concentration of CNPs.

In summary, we have revealed that K⁺ accumatation in cells was related to cell growth induced by CNPs and CNPs promoting uptake through upregulating gene expression in BY-2 cells including NKT1, NHA1 and NtHAK1. K⁺ accumulation reached the maximum value at concentration of 61.25 µg/mL of CNPs. The K⁺ accumulation increased by the addition of exogenous K⁺ (≥ 20 mM) in the presence of CNPs. As well, the carboxyl functional groups on the surface of CNPs were crucial importance for improving K⁺ uptake. The carbonyl of CNPs disturbed by ethanol had no effect on K + uptake.In addition, CNPs may simulate ion channels for K⁺ permeating into cells. The amino acid metabolic effects of CNPs was studied. Accelerating K⁺ accumulation by application of CNPs can open new perspectives for mimics biological channels for ion selectivity. These results supplied mechanism of CNPs promoting plant cells growth for further. Comprehensive investigation of the mechanism of CNPs on plant cell potassium uptake was better for understanding the impact of carbon based nanomaterials on plant cells growth.

Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests Funding There was no funding supported in this paper. Authors' contributions LC, XL and JX conceived and designed the experiments. LC, JY and KL performed the experiments. TL provided the plant cells. LC wrote the paper with support from XP and JX. FX and CN reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements We gratefully acknowledge Xuyue (Beijing) Sci. &Tech. Co., Ltd. for the technical supports.

CNPs:carbon nanoparticles; MWCNTs: multi-walled carbon nanotubes; GO: graphene oxide; SWCNTs:Single-walled carbon nanotubes; NMT:Non-invasive Micro-test Technology; TGA:thermogravimetric analysis; FTIR: Fourier transform infrared spectrum.

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Table 1 Free amino acids analysis treated with CNPs in BY-2 cells.

Retention time	Amino acid	Ctrl	1	2	3	4	5
Retention time	Amino acid	μM/g
22.332	L-Aspartic Acid↑↓	0.267±0.026 c	0.399±0.076 c	0.584±0.020 b	0.762±0.220 a	0.701±0.136 a	0.542±0.150 b
25.499	Hydroxy-L-proline	2.240±0.395 b	1.904±0.446 c	2.646±0.209 a	1.450±0.587 d	2.559±0.558 a	1.787±0.101 c
29.211	L-Threonine↑↓	0.869±0.047 d	1.075±0.056 d	1.334±0.091 c	1.750±0.205 b	1.993±0.328 a	1.412±0.078 c
31.352	L-Serine↑↓	1.917±0.034 d	1.919±0.242 d	2.473±0.163 c	3.013±0.235 a	2.742±0.714 b	2.070±0.266 e
36.416	L-Asparagine	1.204±0.097 c	1.313±0.137 c	1.830±0.126 b	2.125±0.066 a	1.897±0.431 b	1.917±0.880 b
38.967	L-Glutamic Acid	0.217±0.070 c	0.130±0.042 d	0.161±0.024 d	0.439±0.242 b	0.535±0.040 a	0.247±0.129 c
41.108	Glutamine	1.076±0.137 c	0.983±0.293 c	1.373±0.032 b	1.757±0.206 a	1.174±0.409 c	1.746±0.147 a
50.115	L-Proline↑↓	0.360±0.054 e	0.603±0.56 d	1.099±0.115 c	1.612±0.195 a	1.740±0.341 a	1.408±0.268 b
51.567	Glycine↑↓	1.345±0.183 c	1.304±0.217 c	1.690±0.208 b	2.186±0.213 a	2.210±0.416 a	1.651±0.070 b
53.265	L-Alanine	12.522±1.002 a	11.686±1.326 b	11.296±0.313 b	12.703±0.721 a	9.966±1.176 c	6.456±0.258 d
60.425	L-Valine	2.128±0.098 c	2.280±0.249 c	2.850±0.124 b	3.494±0.182a	3.629±0.661 a	2.341±0.313 c
68.309	L-Methionine↑↓	0.326±0.075 e	0.496±0.050 e	0.664±0.021 d	0.767±0.048 b	0.802±0.206 a	0.710±0.192 c
70.909	Cystathionine↑↓	0.904±0.056 f	1.284±0.050 e	1.663±0.114 d	2.362±0.364 b	2.697±0.513 a	1.882±0.085 c
72.709	L-Isoleucine↑↓	2.134±0.083 e	2.625±0.096 d	3.486±0.214 b	3.954±0.236 a	4.152±0.862 a	3.185±0.345 c
73.825	L-Leucine	0.126±0.076 a	0.128±0.049 a	0.130±0.022 a	0.074±0.012 c	0.062±0.014 c	0.096±0.003 b
77.824	N-Leucine↑↓	0.462±0.057 e	0.565±0.009 d	0.664±0.065 c	0.731±0.031 b	0.848±0.145 a	0.679±0.137 c
79.939	L-Tyrosine↓	0.552±0.165 a	0.498±0.168 b	0.514±0.045 b	0.331±0.007 c	0.342±0.110 c	0.300±0.111 c
82.163	β-Alanine↑↓	0.694±0.047 e	0.932±0.070 d	1.297±0.101 c	1.384±0.094 b	1.560±0.221 a	1.236±0.246 c
92.164	γ-Amino-n-butyric Acid	8.992±0.111 d	8.365±1.015 e	9.282±0.472 c	10.189±0.539 b	11.736±0.282 a	5.954±0.238 f
104.221	L-Lysine	0.842±0.049 e	1.193±0.170 d	1.774±0.193 b	1.563±0.850 c	1.253±0.136 d	2.381±0.208 a
106.940	1-Methyl-L-histidine	0.129±0.023 c	0.150±0.046 b	0.159±0.025 b	0.193±0.034 a	0.062±0.008 d	0.096±0.057 d
108.484	L-Histidine↑↓	0.297±0.020 e	0.385±0.038 d	0.541±0.028 c	0.660±0.036 b	0.731±0.121 a	0.511±0.007 c
112.169	3-Methyl-L-histidine	0.126±0.011 d	0.157±0.038 c	0.257±0.023 b	0.163±0.011 c	0.338±0.062 a	0.229±0.014 b
117.503	L-Carnosine	0.144±0.025 b	0.189±0.043 a	0.145±0.096 b	0.133±0.005 b	0.152±0.042 b	0.103±0.022 c
122.311	L-Arginine↑↓	0.737±0.058 d	1.082±0.178 c	1.545±0.114 b	1.693±0.073 a	1.794±0.390 a	1.539±0.371 b

1:treated with 46.88 mM K⁺

2:treated with 46.88 mM K⁺ + 6.25 μg/mL CNPs

3:treated with 46.88 mM K⁺ + 31.25 μg/mL CNPs

4:treated with 465.88 mM K⁺ + 62.50 μg/mL CNPs

5:treated with46.88 mM K⁺ + 125.00 μg/mL CNPs

↑↓Arrows indicated the content of amino acid increased with the concentration of CNPs, and decreased at concentration of 125.00 μg/mL.

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