Salicylic acid nanoparticles (SANPs) improve growth and phytoremediation efficiency of Isatis cappadocica Desv., under As stress

Abstract

Arsenic (As) is a toxic metalloid dispersed in the environment and it cause serious toxicity to plants. Salicylic acid (SA) plays an important role in many plant growth processes involved in plant defence against heavy metal or metalloid stress. In the present study, for the first time, chitosan nanoparticles was synthesised, loaded by SA and the positive role of SA on growth and phytoremediation efficiency of Isatis cappadocica against As toxicity were evaluated. The highest arsenate treatment (1200 µM) caused a decrease in plant biomass that, however, its combined application with salicylic acid nanoparticles (SANPs) substantially increase in shoot height, root length and their biomass production compared with As stressed plants. The pretreatment of SANPs by increasing arsenate supply, simultaneously increased the As concentration in roots and shoots of I. cappadocica which reached a maximum of 705 and 1188 mg/kg, respectively. This results suggest that high levels of As stress induce stress status in I. cappadocica which SANPs pretreatment application limit these toxic effects of As. Therefore, SANPs has a significant beneficial effect on the growth and phytoremediation efficiency of I. cappadocica subjected to As stress.

1 Introduction

Heavy metals and metalloids are naturally occurring elements that have raising concerns over their potential effects on human health and the environment [1, 2, 3]. Some of heavy metals (metalloids) such as arsenic (As), chromium, cadmium, mercury and lead are ranked as public health significance because of their high degree of toxicity [3]. The toxicity of these metallic elements to human health is associated with a number of disease and cause severe organ damage [1, 2, 3]. Therefore the removal of these toxicants from the environment is necessary to maintain green environment.

As is one of the most aggressive metalloid considered as an important environmental contaminant [3, 4]. It is highly toxic to plants and detrimental to their growth and development. As is also able to interfere with chlorophyll biosynthesis through the induction of iron (Fe) deficiency or the inhibition of some key steps of the physiological process [3, 4], which can be detected by reduction of biomass.

Despite the severe symptoms of toxicity appear in many plants species under heavy metal stress, some plants species are able to accumulate and detoxify extraordinarily high levels of heavy metals [4, 5]. In this context, discovery of plants that are hyperaccumulate and tolerate As can be exploited for developing phytoremediation technologies and food security [6, 7, 8, 9, 10].

Among hyperaccumulators, Isatis cappadocica is one of the efficient case in accumulating high As concentration [4, 5, 11], indicating its high tolerance to As. I. cappadocica, as a robust perennial rosette plant, has been established in temperate Asian regions. It can grow in highly impacted As‐contaminated areas and hyperaccumulate As in its areal parts [9].

Plant hormones such as salicylic acid (SA) play an important role in developmental processes, and some of them have key roles in mechanisms leading to increase acclimation to environmental stress. This hormone acting as an important signalling molecule against various biotic and abiotic stresses [12, 13, 14]. Exogenous SA alleviates the toxicity and induces adaptability to heavy metals and metalloids including As [14, 15, 16, 17].

Nanotechnology has emerged as an innovative technology for the elaboration and use of new nanomaterials in the industry and many fields of research [18, 19]. Nanotechnology includes processing, generation, manipulation and deployment of nanomaterials (having one or more dimensions in the order of 100 nm or less) [20]. Nanomaterials hold great promise regarding their application in nutrition and plant protection due to their size‐dependent qualities, high surface‐to‐volume ratio and unique optical properties [21].

Studies showed that nanotechnology has provided the feasibility to enhance nutrient use efficiency in plants [22]. Nanosized formulation of mineral nutrients may improve solubility and dispersion of insoluble nutrients in soil, reduce soil absorption and fixation and increase the bio‐availability of nanostructured particles. Therefore, nanonutrient elements (nanofertilizer) causes use efficiency of nutrients and enhance ability of plants to uptake nutrients [23].

Chitosan, an environmental friendly polymer, is a deacetylated derivative of chitin [24, 25]. Chitosan and its derivatives have been reported to act as a plant growth promoter and elicit natural defence responses in plants [26, 27]. It use as a natural compound for nanoparticle synthesis and encapsulating of fertilizers in agriculture too [28]. Improvement of the ability of plants to absorb nutrients by nanotechnology methods can be a suggestion for use of chitosan nanoparticles loaded by SA to improve resistance of plants to environmental stress. Furthermore, the advantageous role of SA against the abiotic stress, such as heavy metals is well documented in plants. To the best of our knowledge, no study has been attempted so far, elucidating the roles of salicylic acid nanoparticle (SANP) in the growth and As accumulation in roots and shoots of plants, so I. cappadocica was selected due to its hyperaccumulator capacity of As. The present study was done to investigate the change in the levels of growth, As concentration in roots and shoots and translocation and bioaccumulation factors of I. cappadocica, which all contribute to an understanding this plant ability to soil reclamation through the phytoremediation process.

2 Materials and methods

2.1 Materials

Chitosan was purchased from Sigma–Aldrich (USA) with an MW of 60 × 10³ and the degree of deacetylation was 93%. SA was purchased from Merck. Tripolyphosphate (TPP), polysorbate 80 (Tween 80), acetic acid, dichloro methane (DCM) and other reagents were also purchased from Merck.

2.2 Preparation of TPP‐chitosan nanoparticles

Two weight percent chitosan solution was prepared by dissolving it in 1% acetic acid and then polysorbate 80, as an emulsifier (1%, v/v), was mixed into the chitosan solution for 10 min. SA, poorly soluble substance in water, was dissolved in DCM (2:10) and then this oil phase was mixed with aqueous phase (chitosan solution) by probe sonication applying 15 kHz frequency for 10 min. TPP (1%, w/v) solutions were dropped by spray gun into the chitosan solution. Then, nanoparticles were washed with double‐distilled water repeatedly followed by vacuum drying for 12 h [29, 30]. The above synthesis process is explained in Fig. 1.

Fig. 1.

Synthesis process of TPP‐chitosan nanoparticles

2.3 SA content of nanoparticles

SA loading was determined by ultracentrifugation of nanoparticles solution in 40,000 rpm for 45 min, and then the amount of SA in supernatant was measured using spectrophotometer (Agilent 8453) at 225 nm. Loading efficiency (DL) % can be calculated as follows [3]:

$$\begin{array}{rl}\mathrm{DL}\text{\%}& =[(\mathrm{total}\mathrm{amount}\mathrm{of}\mathrm{salicylic}\mathrm{acid}-\mathrm{free}\mathrm{amount}\mathrm{of}\mathrm{salicylic}\mathrm{acid})/\\ & \mathrm{total}\mathrm{amount}\mathrm{of}\mathrm{salicylic}\mathrm{acid}]\times 100.\end{array}$$

2.4 Morphological characterisation of salicylic‐loaded chitosan nanoparticles

To capture the transmission electron microscopy (TEM; Zeiss‐EM 10C‐Germany) microphotographs, the nanoparticle samples stained with 2% (w/v) phosphotungstic acid were employed. Investigation of surface morphology of nanoparticles was performed by scanning electron microscopy (SEM; KYKY‐EM3200, China). Size and zeta potential were measured by photon correlation spectroscopy (PCS; Nano ZS4700 Nanoseries, Malvern Instruments, UK).

2.5 Plant material and growth conditions

Seeds of I. cappadocica Desv. were collected from a population growing at the gold‐As Zarshuran deposit mine, with grid reference of 36°43′04″ N 47°08′02″ E, 40 km north of Takab in the West Azarbaijan province, northwest of Iran. Collected I. cappadocica seeds were surface sterilised in 50% (v/v) commercial bleach (4% NaOCl) for 2 min, followed by rinsing three times for 5 min in sterilised distilled water. The seeds were germinated on sterile distilled water moistened filter paper in petri dishes for 4 days at room temperature, <25°C. After germination, seedlings were then transferred to 1 l polyethylene pots (four seedlings per pot) immersed in a nutrient solution [5] composed of 0.5 mM KNO₃, 0.75 mM Ca (NO₃)₂, 0.5 mM KH₂ PO₄, 0.2 mM MgSO₄, 15 µM H₃ BO₃, 2 µM MnCl₂, 1 µM ZnSO₄, 0.5 µM CuSO₄, 0.5 µM Na₂ MoO₄, 2H₂ O and 50 µM Fe‐EDTA (pH 6.0). The nutrient medium was continuously aerated with an aquarium air pump, renewed twice per week and adjusted at the daily pH levels of 5.5 ± 0.1 with HCl or NaOH. The seedlings were pretreated with 250 µM of SANPs (SA loaded chitosan nanoparticle) for 10 days. After that plants were exposed to different concentrations of arsenate (0, 400, 800 and 1200 μM), for another 21 days. Arsenate doses were chosen based on levels occurring in nutrient solution where I. cappadocica plants have been found on the growing site [5]. Plants were grown in a growth chamber (Conviron model CG72) with 14/10 h light/dark cycles; the temperature was kept at 26°C during the day and 20°C during the night. Light intensity was ∼280 µmol m⁻² s⁻¹. Each treatment was replicated three times and each time, the pots were randomly arranged during the growth period.

2.6 Measurements of growth parameters

Plants were harvested after they were exposed to arsenate for 21 days and were rinsed with tap water. The fresh weight was determined immediately after harvesting and length of root and shoot were measured with the help of a scale.

The stress tolerance index (TI) (%) was calculated as

$$\begin{array}{rl}\mathrm{tolerance}\mathrm{index}& =\mathrm{shoot}\mathrm{length}\mathrm{of}\mathrm{stress}\mathrm{plant}/\\ & \mathrm{shoot}\mathrm{length}\mathrm{of}\mathrm{control}\mathrm{plant}\times 100\end{array}$$

2.7 Determination of As concentration

Total As was determined in acid digested of ground shoot (100–200 mg DW) mixed with 2 ml of HNO₃ (67% suprapur) and 2 ml of H₂ O₂ (30% by volume), and then microwave digested at 95°C. The digest was diluted with a solution containing 10% HCl, 5% ascorbic acid and 10% KI, and then analysed by using a hydride generation–atomic absorption spectrometry with a flow injection hydride generator interfaced with a Shimadzu AA‐6200 atomic absorption spectrometer (HG‐AAS). Reference standard for calibration of the AAS was made using 1000 mg l⁻¹ (Beech leaves material FD8, Commission of the European Communities, Joint Research Centre ISPRA).

2.8 Translocation factor

As translocation from shoot to root was measured by translocation factor (TF) which is given as

$$\mathrm{TF}=C_{\mathrm{shoot}}/C_{\mathrm{root}}$$

C _shoot and C _root are metals concentration in the shoot (mg kg⁻¹) and root of plant (mg kg⁻¹), respectively.

2.9 Bioaccumulation factor

Bioaccumulation factor (BF) was calculated by

$$\mathrm{BF}=C_{\mathrm{root}}/C_{\mathrm{medium}}$$

C _root /C _medium are metals concentration in the plant root (mg kg⁻¹) and medium concentration (mg kg⁻¹), respectively.

2.10 Statistical analysis

The data represent mean calculated from three replicates ±standard error (S.E.). One‐way ANOVA was employed to confirm the variability of data and validity of results with different rates of As addition. Duncan's multiple range test was employed to determine the significant difference between treatments to a significance level of P  < 0.05 or very significant as P  < 0.001. Statistical analyses were performed using SPSS (20) software.

3 Results and discussion

3.1 Characterisation of chitosan nanoparticles

TEM image of the samples containing SA loaded chitosan nanoparticle is shown in Fig. 2. SEM image showed spherical shape and smooth surface with a particle size in nanometric scale (Fig. 2). Fig. 4 shows a narrow size distribution with a poly dispersity index of 0.232, indicating chitosan nanoparticle mono dispersity, with an average diameter of 190 nm. The particle size of SA‐loaded chitosan nanoparticle in SEM images is in agreement with the results obtained by TEM (Fig. 2) and the result of PCS (Fig. 3). All applied techniques are indicative of circular shape and no aggregation of the nanoparticles. The average diameter determined by both SEM and TEM was found to be ∼200 nm which is approximately the same as that of obtained by PCS.

Fig. 2.

(a) TEM image of salicylic loaded chitosan nanoparticles and (b) SEM image of salicylic‐loaded chitosan nanoparticles

Fig. 4.

Effect of As and SANPs (SA loaded chitosan nanoparticle) on

(a) Length of shoot, (b) Length of root of I. cappadocica. Values represent mean ±SD. Different letters indicate significant differences at the 5% level according to the Duncan test

Fig. 3.

Size distribution of salicylic‐loaded chitosan nanoparticles

3.2 Growth parameters

As is well known to adversely affect the plant growth [5, 31]. Growth inhibition is one of the important manifestations of As‐induced toxicity in plants [32, 33, 34].

SA is an important signalling molecule in plants and induces plant tolerance to various biotic and abiotic stresses [35]. It is reported that exogenous application of SA ameliorates the damaging effects of heavy metals, such as cadmium [14, 15, 36, 37], lead and mercury [38, 39]. Also, increasing evidences indicate that SA is involved in plant detoxification of heavy metals [40, 41, 42, 43]. Therefore, the present study was performed to investigate the mechanisms of the beneficial effect of SANPs on I. cappadocica exposed to toxic arsenate levels. The growth of the plants exposed to arsenate with pretreatment SANPs was determined in terms of the effects on length and weight of shoots and roots (Figs. 2 and 3). Preliminary experiments with SA pretreatment were carried out to determine the effect of SA, which cause stimulation of growth (data not shown). Based on these early tests, further experiments were carried out for SANPs pretreatment.

The length of shoots and roots were decreased with increasing As stress with the greatest reduction observed at the highest arsenate level (Figs. 4 and 7 a). It was clear that the most negative effect of As stress was observed on the reduction of length of shoots and roots. At arsenate treatment of 1200 µM, the reductions of shoot length were 26% (Fig. 4 a), while it was 36% for root length (Fig. 2 b) which is showed the more sensitivity of roots to toxic levels of As. In contrast, SANPs significantly enhanced the length of shoots and roots (Figs. 4 and 7 b).

Fig. 7.

Effect of As‐stress and SANPs pretreatment on growth of I. cappadocica plants. The growth decreases of plants exposed were compared with

(a) 1200 μM As, (b) Pretreatment of SANPs, which indicate significantly improved growth in this treatment

The shoot and root fresh weight was decreased proportionally with increasing arsenate concentration and 1200 µM arsenate caused 27 and 44%, respectively, reduction in the values of this parameter (Fig. 5). However, SANPs pretreatment seemed to restrict reductions in biomass (fresh weight), diminishing the effects of As. Indeed, pretreatment of SANPs increased fresh weight significantly both in roots and shoots (Fig. 5).

Fig. 5.

Effect of As and SANPs (SA loaded chitosan nanoparticle) on

(a) Fresh weight of shoot, (b) Fresh weight of root of I. cappadocica. Values represent mean ±SD. Different letters indicate significant differences at the 5% level according to the Duncan test

Plant biomass production and plant elemental uptake are two key factors for successful application of phytoremediation [44]. Applied SA was reported to improve biomass in several plants heavy metal exposed [43, 45, 46, 47]. Also, SA pretreatment alleviates As toxicity and increase growth in Arabidopsis thaliana [48], Matricaria recutita [49] and Oryza sativa [12].

The effect of As on plant shoot TI is presented in Fig. 6. According to the results, I. cappadocica is tolerant to high arsenate concentrations, as shown by its TI values (Fig. 6). SANPs pretreatment caused obvious TI increase (>140%) as valuable parameter for phytoremediation (Figs. 6 and 7). Internal detoxification of As by SANPs must be an important feature of this hyperaccumulator species. This threshold value is similar to that reported by Guo et al. [36, 50], Mostofa and Fujita [41], Li et al. [51], who grew the plants on a medium applied with SA under different heavy metal stress.

Fig. 6.

Effect of As and SANPs (SA loaded chitosan nanoparticle) on TI of I. cappadocica. Values represent mean ±SD. Different letters indicate significant differences at the 5% level according to the Duncan test

3.3 As accumulation

The total amount of As accumulated in roots and shoots of I. cappadocica is shown in Figs. 8 and 9. As shown in the figures, the concentration of As in the plant increased with increasing concentration of arsenate in the medium. Salicylic nanoparticles had augmenter effects on As uptake in the roots and As concentration in the roots was greater than in shoots (Figs. 8 and 9). When arsenate treatment was up to 1200 µM, the accumulation of As in roots increased markedly. However, it was no significant effect on the accumulation of As in the shoot (Fig. 9). Indeed, SANPs pretreatment was more effective in increasing As accumulation in roots than that of shoots. Thus, SANPs have negatively impacted on the root to shoot translocation of As. Current results agree with previous investigations reporting that addition of SA caused more accumulation of metals in roots than in shoot [17, 52].

Fig. 8.

Effect of As and SANPs on As concentrations (mg kg⁻¹, DW) in roots of I. cappadocica. Values represent mean ±SD. Different letters indicate significant differences at the 5% level according to the Duncan test

Fig. 9.

Effect of As and SANPs on As concentrations (mg kg⁻¹, DW) in roots shoots of I. cappadocica. Values represent mean ±SD. Different letters indicate significant differences at the 5% level according to the Duncan test

3.4 Phytoremediation efficiency

The BF and TF were used to investigate the heavy metal uptake and bio‐concentration behaviour [53]. Both BF and TF can be used to estimate a plant's potential for phytoremediation purposes [54]. BF > 1.0 was found in heavy metal‐accumulating plants, whereas they were typically <1.0 in heavy metal‐excluding plants [55]. TF > 1.0 indicates that the plant is effective in the translocation of heavy metals from the root to the shoot tissue [56, 57]. The effects of arsenate and SANPs pretreatment on TF and BF of I. cappadocica are given in Table 1. TF values of I. cappadocica in 800 and 1200 µM arsenate treatments were >1, while SANPs pretreatment decreased TF in all arsenate levels (Table 1). The BFs of all treatments, except 800 and 1200 µM As, were >1.0 (Table 1). BF of As + SANPs ranged from 1.02 to 2.27, suggesting considerable bioaccumulation of As in roots. This suggested that I. cappadocica had brawny tolerance and hefty accumulation potential for As. Moreover, the elevated TF demonstrated that the plant is not only capable to tolerate As but can also translocate it to the shoots. The stimulatory effect of SANPs was seen on higher As accumulation in roots than in shoots of I. cappadocica, when it was exposed to As. It seems to be SANPs help plant roots to build efficient barriers restricting As entry into the xylem, and thus reducing its translocation into shoots. This performance is one of the several strategies of tolerance to As by plants. In similar case, the same positive effect of SANPs on growth parameters was seen in I. cappadocica (Table 1 and Figs. 4 and 6). The highest and the lowest BFs were observed in 400 µM As + SANPs and 800 µM As‐SANPs, respectively (Table 1). Generally, SANPs pretreatment resulted in a remarkable increase in the BF and growth parameters for phytoremediation efficiency of As. There were no literature on SA effect in an increase in the BF by hyperaccumulator plants. Based on presented results, it could be conclude that SANPs may help in metal uptake by chelating As in the solution and then release As in the plant tissues.

Table 1.

Translocation factor and bioaccumulation factor of I. cappadocica exposed during 21 days to different concentrations of As with and without SANPs pretreatment

Arsenate treatments, µM	SANPs, µM	TF	BF
400	0	0.679 ± 0.015	1.28 ± 0.084
400	250	0.425 ± 0.028	2.27 ± 0.36
800	0	1.274 ± 0.044	0.76 ± 0.028
800	250	0.794 ± 0.19	1.02 ± 0.15
1200	0	1.096 ± 0.024	0.78 ± 0.073
1200	250	0.545 ± 0.010	1.08 ± 0.033

4 Conclusions

Since the growth parameters are a very important factor for the success of the hyperaccumulator plants, this study was undertaken to investigate the influence of SANP on improving growth and enhancing I. cappadocica tolerance to As toxicity. Our results shed new insights into the pretreatment of SANPs on growth parameters in I. cappadocica. In overall, the results of this study signify the role of SANPs in regulating the As stress response of I. cappadocica and suggest SANPs could be used as a potential growth regulator to improve plant growth and phytoremediation efficiency under As stress. In SANPs pretreated plants, As accumulation in shoots is relatively lower when compared with roots. Since roots are coming in direct contact of As and SANPs, it shows the aggregation on the root surfaces. Other possible mechanisms, for instance, the alterations in antioxidant responses after pretreatment of SANPs in roots and shoots, need to be further investigated.