Bismuth vanadium oxide can promote the growth and activity of roots in Arabidopsis thaliana

The excellent properties of nanomaterials have been confirmed in many fields, but the effects on plants are still unclear. In this study, we have found that bismuth vanadium promoted the growth of taproots and the development of lateral roots, increased the roots activity by regulating the expression of polyamine or hormone-related genes and reducing the accumulation of reactive oxygen species in Arabisdopsis thaliana. Meantime, BV had higher antimicrobial effect than that of graphene oxide (GO). These results showed us a new perspective possibility of nanomaterials assisting the development of plants and crops, which is expected to become one of the ways to solve the problem of controlling and promoting the development of plants and crops.


INTRODUCTION
Compared with traditional materials, nanomaterials have many advantages, and are applied in the fields of information industry, environmental industry, energy and environmental protection, biological medicine and other fields [1]. The growth and development of plants are regulated by many factors, such as temperature and hormone [2][3][4][5][6]. Meanwhile, more and more studies show that nanomaterials also have an impact on the growth and development of plants. For example, modified polystyrene nanomaterials with different polarity could inhibit the roots development of Arabidopsis [7]. Copper oxide nanoparticles regulated the phenotype of mung bean [8]. CuONPs can decrease the mitotic index of onion roots tip gradually and increase the abnormal index [9]. Furthermore, the effects of nano-TiO2 foliage intervention on cadmium bioaccumulation, stress kinase and potential dietary health risks in cowpea plants were also reported [10]. What's more, the lignification degree of xylem in roots and stems of fenugreek treated with nanosilicon materials was significantly higher than that of control plants [11]. Most of the nanomaterials above showed toxic effects on plants, however, some of them showed positive role. A research reported that graphene oxide (GO) promoted the growth of watermelon, including increasing roots length, leaf area, leaf number and flower bud formation [12]. Multi-walled carbon nanotubes (MWCNTs) could stimulate the seed germination of three important crops (barley, soybean and maize), enhance the root length in the seedlings of Phaseolus mungo and the germination index of Brassica juncea in low concentration [13][14][15].
However, its application in plant related fields is still less, and due to the variety and different characteristics of nanomaterials, it also shows different effects on plants.
Metal vanadate have been widely used for potential applications as batteries, implantable cardiac defibrillators, and photo catalysis [16]. Specially, bismuth vanadate nanomaterials (herein after referred to as BV) has emerged as a promising candidate due to its unique nontoxicity, chemical stability, optical, and ferro-elastic properties [17]. Various applications of BV has been well studied in eco-friendly yellow pigment， water splitting, sensors, pollutant degradation. Recently, BV has been drawn great attation in biological applications. The BV composite material exhibited excellent potential for inactivation of Escherichia coli [18][19][20]. These works provided evidence about the positive effects of the BV nanomaterials on microbiome. However, currently no or little attempts have been made to explore the effect of BV nanomaterials on plants. Therefore, the objective of this study was to use Arabidopsis thaliana as a model plant to determine the effect of BV on some aspects including the length of taproot, the number of lateral roots, the activity of plant root and superoxide anion (O2 .-) generation, the antimicrobial activity and the expression of related genes ( Figure 1). This evaluation was based on the effects of applying different concentrations of the BV on root elongation rate, so as to clarify the effect of BV on root in Arabidopsis thaliana.

Plant material
Seeds of Arabidopsis thaliana L., Columbia-0 (Col-0) were surface-sterilized with 70% ethanol and 20% bleach. Materials were grown at 22 ℃/20 ℃ under a photoperiod with 16 h of light and 8 h of dark and a light intensity of 100 μmol·m −2 ·s −1 conditions in an incubator (QY-14; Nanjing Quanyou Electronic Technology Co., Ltd, China).

Preparation and characterization of BV
Bi(NO3)35H2O (2.1830 g) and EDTA (4 g) were dissolved in dilute HNO3 solution (50 mL, 2 mM), and stirred at 90 C for 30 min (200 rpm) to get Solution A. NH4VO3 (0.5260 g) was dissolved in deionized water (50 mL) at 60 ℃ to obtain Solution B. Then, solution B was mixed with solution A, and the pH was adjusted to 7 by NH4OH. The above mixed solution was stirred for 1 h at 50 ℃, poured into a 150 mL Teflon-lined stainless-steel autoclave, and maintained at 180 ℃ for 6 h. The prepared precipitate was washed with ethanol and deionized water for several times, and dried in a vacuum oven at 75 ℃ for overnight to formate BV nanomaterials.
Sample morphology and surface elemental composition was examined by scanning electron microscopy (SEM; JSM6510, JEOL, Japan) coupled with energy-dispersive spectrometry (EDS) at an accelerating voltage of 10 kV. X-ray diffraction (XRD; Ultima IV, Rigaku, Japan) patterns of samples were recorded by using high-intensity Cu Ka radiation (=0.154 nm) in the range of 2 =10-80 at a scan rate of 5 ·min -1 .

Preparation of roots for analysis
With respect to plants grown in solid media, A. thaliana seeds were germinated on a square plate (10 cm × 10 cm) that contained sterilized solidified half-strength MS (Sigma-Aldrich, St. Louis, MO, USA) media that consisted of 0.8% agar (Affymetrix, Inc. Cleveland, Ohio, USA) and 1% sucrose. 10 g BV powder was dissolved in 1 L of deionized water and sterilized in an autoclave (SANYO Labo Autoclave, MLS-3020). BV solution that had been sonicated for 1 h was blended with the sterilized half-strength MS medium by stirring with 0, 20, 50, 100 and 200 µg·mL -1 nanomaterials, and the resulting medium (here in after referred to as the BV/MS medium). Totally 30-40 seeds were planted in the BV/MS medium and placed in 4 ℃ for two days and then transferred to a growth chamber as described above.
The roots were imaged with a scanner, and the primary root length was measured using ImageJ software (National Institutes of Health, United States) after six days of exposure. The lateral root number was analysed after eleven days of exposure. The roots of the plants exposed to nanomaterials for 6 days were stained with propidium iodide for viability testing meristem, extension zone length, tip diameter and rootcap size of primary root as described previously [21].

Detection of superoxide anion radical O2 .− and root activity
To visualize O2 .− and root activity in plant roots in situ, nitroblue tetrazolium (NBT) and 2,3,5-Triphenyl tetrazolium chloride (TTC) staining were performed, respectively, as described by previously [22,23] and modified slightly. The root O2 .− content was measured by incubating the plants in 1% NBT within 20 mM K-phosphate, then washed with distilled water, decolorized with 70% ethanol solution in 90 ℃ water for 20 min. The root activity was measured by incubating the plants in 2% TTC at 37 ℃ for 5 h. Images of the plants or roots were obtained under brightfield illumination.

Antimicrobial effect of nanomaterials
In order to clear the antimicrobial effect of nanomaterials, the seeds of A.
thaliana were cultured in half-strength MS media with adding 0, 50, 100, 200 µg·mL -1 non-autoclaved nanomaterials and analysed the microbial number after 4 d. (At3G18780) was used as a reference gene. The primers used for qPCR are listed in Table S1.

Statistical analyses
Without special instructions, all experiments were repeated at least three times. SPSS 20.0 and SigmaPlot 10.0 were used for statistical analysis and plotting.
Statistical differences were analysed by Duncan test. The data were considered significant in accordance with the following criteria (P < 0.05).

Fabrication and Characterization of nanomaterial
The morphology and structures of the as-prepared BV nanomaterials were detected by SEM (Figure 2). Geometrically shaped-like nanoparticles are observed for BV ( Figure 2A). In addition, elements of C, O, Bi, and V were clearly displayed in the the EDS spectra ( Figure 2B

Effects of nanomaterial on Arabidopsis roots
To confirm the influence of BV on the primary root length and lateral root numbers, these two traits were evaluated after 6 and 11 days of plant growth on the various BV/MS media. Figure 3 shows that nanomaterial has effects on the length of primary roots in Arabidopsis, and the different concentration of nanomaterials had inconsistent changes. Compared with the control, 20, 50 and 100 µg·mL -1 BV significantly reduced the length of taproot about 13.0～20.0%, while 200 µg·mL -1 BV remarkably enhanced the length about 49.0%. This may imply that BV has a dual effect on the length of plant roots. This phenomenon of dual effects on plants is not the first time, and a report showed that GO had a similar phenomenon [12]. There may also be another situation, that is, it is related to the concentration of BV. This hypothesis is based on the fact that BV is not detected on the surface of the treated roots observed by EDS ( Figure S1). This implies that BV plays a role after entering  In addition, we also observed that the addition of nanomaterial BV can also affect the lateral roots number (Figure 4). BV treatment of different concentration increased the number of lateral roots of all the plants, and the difference of 20 µg·mL -1 and 50 µg·mL -1 treatments was at a significantly level. There was no significant difference among the other treatments compared to the treatment without adding nanomaterial. In general, the effect of low concentration was more beneficial to increase the number of lateral roots. Previous studies have shown that the development of lateral roots can be affected by many kinds of plant hormones [24]. In our study, the effect of nanomaterial BV on lateral roots may be achieved by changing plant hormones. The lateral root is connected with the taproot, and the water and nutrient can flow through the catheter and sieve tube. Generally speaking, the growth of lateral roots was inhibited by taproot, especially near the root tip.
To a certain extent, BV treatment was in line with this trend. It is necessary to further study these phenomena, and the cause of these different influence modes can increase our understanding of how BV affects plants.

The root activity and O2 .− production analysis
The above experiments had shown that BV promoted roots development in plants. Therefore, we tried to make clear whether root activity and O2 .− also changed.
O2 .− belongs to the reactive oxygen species group, which has strong oxidizability. It plays an important role in the process of physical reaction [25]. When organisms are stressed, the production of reactive oxygen species in the body will increase greatly; when it exceeds the antioxidant defense capacity of organisms, cellular components such as lipids, proteins, nucleic acids and so on will be irreversibly damaged under the action of O2 .− to interfere with cell metabolism or cause cell death [26]. Succinic acid is a key intermediate in the tricarboxylic acid cycle. Under physiological conditions, succinate dehydrogenase in mitochondria oxidizes succinate to fumaric acid and releases electrons to participate in oxidative phosphorylation [27]. The reduction of TTC indicates the activity of dehydrogenase can be used as an indicator of root activity. Consequently, TTC and NBT staining were performed ( Figure 6).
As a method to identify roots vitality, TTC staining is based on the principle that living tissue can produce hydrogen ions under the action of dehydrogenase and has reduction ability. The depth of tissue coloration indicate the strength of root activity.
After growing in half-strength MS medium for 6 days, we found that the roots tip color of 200 µg·mL -1 BV was significantly darker than that of the control, indicating that the seedlings treated with appropriate concentration of nanomaterials increased the roots activity of Arabidopsis. Xie et al also reported that plant root activity and the plant root growth were positive related under GO treatment in napus seedlings [28].
This suggests that BV can affect the tricarboxylic acid cycle and plays a positive role in enhancing plant activity.
Superoxide dismutase (SOD) is an enzyme that scavenges O2 -. O2can reduce NBT to blue methylhydrazone, so SOD inhibits the formation of methylhydrazone.
The deeper the blue color of the tissue treated with NBT solution, the lower the enzyme activity. Conversely, the higher the enzyme activity. Therefore, this method can be used to measure O2 -. In this experiment, we found that Arabidopsis seedlings of 200 µg·mL -1 BV was slightly lighter than that of the control, indicating that the accumulation of O2in seedlings was reduced by appropriate concentration of BV. The plants respond to nanoparticles mediated stresses through reactive oxygen species generation [29]. It is widely accepted that the toxicity of nanoparticles on plants is commonly evident at high concentrations and attributed to the generation of reactive oxygen species (ROS) [30]. But a report showded that SOD activity was widely stimulated after the exposure of plants to TiO2 NPs and reduced the level of total ROS. Our data are consistent with the results [31]. It suggested that BV enhance the SOD activity and promoted the plant growth by inducing the accumulation of ROS.

Antimicrobial effect of nanomaterials
In view of the reported antimicrobial activity of BV [32], the antimicrobial experiments of BV were done (Figure 7). Graphene oxide (GO) with certain antimicrobial activity [33][34][35] was selected as the control to compare the antimicrobial effect of BV. Adding different concentrations of nanomaterial unsterilized BV into half-strength MS medium, it was found that the infection degree of BV was lower than that of GO after 4 days of culture. There was no significant difference between 100 and 200 µg·mL -1 BV treatment, but both were significantly higher than 50 µg·mL -1 treatment. This indicates that BV has certain antimicrobial activity. However, with the increase of GO concentration, the species, number and size of infected microbe increased significantly (Figure 7). It can also be seen from the figure that the growth of Arabidopsis in the medium added BV is better than that in the medium added GO. These results indicate that the two nanomaterials have different resistance to microbe, the antimicrobial activity of BV was better than that of GO

Changes of gene expression related to root development in Arabidopsis
Now that we have found nanomaterial BV can change root length and lateral root number, some genes related to root development are also further investigated. Therefore, we selected three types of genes, among which two genes mainly affected the primary roots (ADC1, DAR2), two genes mainly affected the lateral root ( ARF19, CKX1), and two genes affected both (ERFⅡ, IQM3) ( Figure 8). Surprisingly, in the primary roots, ADC1, DAR2, ERFⅡ and IQM3 show the same trend. ADC1 can affect the synthesis of polyamines in Arabidopsis [36], and studies have shown that polyamines can regulate root length [37]. DAR2 can be associated with a variety of hormones, and then affect the development of root meristem [38]. ERFⅡ, as a transcription factor, is also regulated by hormones, thus affecting the development of root [39]. In Arabidopsis, IQM family belongs to calmodulin binding protein family with IQ motif. IQM3 is involved in the regulation of plant root development [40].
After treatment with BV, ERFⅡ and IQM3 were significantly up-regulated. In general, the expression levels of the four genes were significantly up-regulated. These results suggest that BV may be involved in the regulation of plant taproot development in a variety of ways.
Furthermore, in the lateral root, the related genes show a more complex phenomenon. ARF19, as a gene mediating hormone regulation of lateral roots, is the most in-depth and clear regulatory pathway [41]. In the experiment, BV treatment increased significantly the expression of ARF19. CKX1 could mediate cytokinin to regulate lateral root development, which was significantly down-regulated after BV treatment. Moreover, IQM3 shows a similar level between BV treatment and the control. ERFⅡ was significantly up-regulated in root treated with BV. All kinds of signs showed that BV can regulat root development by mediating hormones. What's more, BV showed better antimicrobial activity than GO. In addition, it proved that the expression level of root development related genes also changed. In conclusion, proper concentrations of BV are expected to be used as promoters for plant growth and development. BV is expected to be widely used in plant related fields due to its excellent properties, low production cost and antimicrobial properties.
However, there are many problems to be solved. For example, only Arabidopsis is used as experimetal materials in this research, whereas how BV affects other plants remains to be explored. Finally, it is necessary to explore the mechanism of nanomaterials affecting plants, and deepen the understanding of them.

Supplementary Materials
Table S1 : Primers used for qRT-PCR Figure S1: Effect of nanomaterial BV on the extension zone of taproot in Arabidopsis.