Exogenous addition of nitrate nitrogen regulates the uptake and translocation of lead (Pb) by Iris lacteal Pall.var.chinensis (Fisch.) Koidz.

SUN Mengjie, GUO Shiwen, XIONG Chunlian, LI Pinfang*

1 College of Land Science and Technology, China Agricultural University, Beijing 100193, China;

2 College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China;

3 Yibin Ecological Environment Monitoring Center, Sichuan 644099, China

Abstract: Since Pb is a non-biodegradable inorganic pollutant and a non-essential metal, its long-term presence in soil poses a great threat to the environment.Iris lactea Pall.var.chinensis (Fisch.) Koidz., a perennial dense bush herb with high resistance of Pb and wide adaptability, was used in pot experiments to study the effects of exogenous nitrate N (-N) on the absorption and transportation of Pb and plant growth under different Pb concentrations.Then, the mechanism of -N affecting Pb and nutrient uptake and transport was explored.The concentration of Pb in the experiment ranged from 0 to 1600 mg/kg, and the added concentration of -N was 0.0-0.3 g/kg.The results showed that I.lactea was highly tolerant to Pb, and the shoot fraction was more sensitive to varied Pb concentrations in the soil than the root fraction.This protective function became more pronounced under the condition of raised Pb concentration in the soil.When the concentration of Pb in the soil reached 800 mg/kg, the highest Pb content of I.lactea was found under the condition of 0.1 g/kg of -N addition.When Pb concentration in the soil increased to 1600 mg/kg, the increase in -N addition promoted Pb uptake by the root.To ensure the well growth of I.lactea and the effect of remediation of Pb-contaminated soil, the recommended concentration of -N in the soil is 0.1 g/kg.This result provides a theoretical basis for exogenous N regulation of phytoremediation of Pb-contaminated soil.

Keywords: Iris lactea; nitrate nitrogen; plant nutrient; lead accumulation; absorb; transport

1 Introduction

Lead (Pb) pollutants can enter soil environment through various ways, causing severe soil pollution (Sinha et al., 2006).Over the past 50 a, approximately 7.83×105t of Pb has been discharged into the global environment, resulting in varying degrees of Pb pollution in the soil around the world (Duzgoren-Aydin, 2007).Since Pb is a non-biodegradable inorganic pollutant as well as a non-essential metal, its long-term persistence in soil is a great threat to the environment(Amari et al., 2017; Gerhardt et al., 2017).Excessive Pb enters the environment and participates in the water-soil-biological system cycle.It accumulates in the roots, stems, leaves, and seeds of plants through the absorption of plants, and endangers the health of animals and human beings through the enrichment of food chain (Salazar et al., 2016; He et al., 2019).Pb is highly toxic and hazardous to humans as well as plants even at low concentrations.The excessive amount of Pb in soil may cause stress-induced changes in plants including growth reduction, decreased biomass,leaf chlorosis, and other physiological and biochemical changes (Chandana et al., 2019).

Pollution remediation methods of heavy metal in soil mainly include physical methods (e.g.,landfilling and replacement with uncontaminated soil), and chemical methods (e.g., chemical fixation, leaching, and extraction) (Park and Son, 2017; Liu et al., 2018).The above methods are inefficient and costly to operate, and may also cause damage to the original soil biological environment.Due to the decreased mobility of Pb in soil, tolerant plants may concentrate Pb in their roots.Studies have shown that the ability to translocate Pb into the aboveground parts may be very low (Salazar and Pignata, 2014).In this context, testing of metal tolerant plant species whether capable of accumulating Pb from soil into their roots or translocating into shoots becomes highly significant.In recent years, in situ remediation of contaminated soils using heavy metal hyperaccumulator plants has been accepted as very promising method.The phytoremediation method is characterized by the high efficiency, no secondary pollution,long-lasting effect, low cost, and easy application (Chaney et al., 2005; Han et al., 2008).For example, Helichrysum microphyllum subsp.tyrrhenicum is an endemic plant species in Sardinia and Corsica, Europe, with good tolerance to heavy metals such as zinc (Zn), Pb, and cadmium(Cd).It was used as a pioneer species for mine soil remediation by Boi et al.(2021).

Several exogenous substances can affect the uptake and transport of heavy metals by plants.The most studied exogenous substances are chelating agents and fertilizers (Vassil et al., 1998;Shen et al., 2002).Nitrogen (N) fertilizer mainly affects the activity of heavy metals through the acidification and alkalization effects of NH+4-N and NO-3-N in the rhizosphere (Zeng et al., 2020).When NH+4-N is absorbed, the plant secretes H+, causing acidification around the rhizosphere;while absorbing NO-3-N, the plant secretes OH-, causing alkalinization of the rhizosphere(Wallace, 1979).Pb contents in the roots and shoots of maize treated with NH+4-N were significantly higher than those treated with NO-3-N, with an increase of about 20%.And the application of NH+4-N fertilizer can increase the content of Pb in plant tissues (Lou et al., 2005).Some studies have pointed out that NO-3-N can promote the uptake and transport of heavy metals by plants.Nitrate fertilization has been shown to increase Zn hyperaccumulation in Nocceaea caerulescens (J.Presl & C.Presl) F.K.Mey (Schwartz et al., 2003; Monsant et al., 2008; Xie et al.,2009).

I.lactea is a perennial herb that is distributed in the northern temperate regions of Asia, Europe,and North America.It also has a wide distribution in northwestern, northeastern, and northern China.Simultaneously, being a long-term growth and ornamental plant, I.lactea has strong tolerance and uptake capacity for Pb (Bai et al., 2008; Ayyasamy et al., 2009).Suzhen et al.(2018)found that the content of Pb in the shoots (leaf and stem) of I.lactea was 983 mg/kg, and the transport coefficient was greater than 1, indicating it has the potential to accumulate high amounts of Pb in its tissues (Yuan et al., 2018).Han et al.(2008) speculated that I.lactea can sacrifice part of its cells to adsorb Pb, thus reducing the toxicity of Pb and ensuring the normal growth and development of the plant.The results of hydroponic tradition showed that Pb contents in the aerial part and root of I.lactea were 1109 and 2408 mg/kg, respectively, exceeding the standard for hyperaccumulators (1000 mg/kg).Thus, the application of I.lactea to remediate Pb-contaminated soils is feasible (Han et al., 2013).However, the remediation capacity of I.lactea under exogenous addition of fertilizer based on soil culture has apparently not been investigated, while previous studies on the enrichment of I.lactea for Pb were hydroponics.Under different culture conditions, Pb accumulation and translocation to the shoots, accumulation mode, and detoxification mechanism of plants were relatively different.Accumulation of Pb in I.lactea under soil cultivation was significantly reduced compared with that under solution culture condition, especially in the shoots.The reason for this may be related to the low water solubility of Pb and the less available Pb in soil that can be uptaken by plants (Zhuang et al., 2000).In conclusion, NO-3-N can significantly promote the uptake of heavy metals by plants, but the remediation potential of I.lactea in Pb-contaminated soils remains unclear.In the present study,the effects of exogenous-N on the absorption and transportation of Pb, nutrient accumulation,and growth of I.lactea under different Pb concentrations were studied.The result might provide sustainable management and remediation for Pb-contaminated soils.

2 Materials and methods

2.1 Plants and soil sampling

I.lactea seeds and soil samples were obtained from the Yanqing Experimental Base of Beijing Oasis Technology Co., Ltd., China.Soil samples were sieved to a particle size of ＜2 mm to exclude the coarse and large debris, and were air-dried (22°C) for 1 week.Soil properties are shown in Table 1.

Table1 Physical-chemical properties of the soil

2.2 Pot experiment

Plants were grown in the plant cultivation room (25°C (±1°C)/12 h light, 22°C (±1°C)/12 h dark,and 63% relative humidity) of the Department of Soil and Water Sciences, School of Land Science and Technology, China Agricultural University.After disinfection with 3% H2O2and repeated washing with deionized water, I.lactea seeds were mixed with river sand sterilized at 105°C and refrigerated at 4°C.After two months, seeds were disinfected with 0.5% NaClO for 20 min.After washing with tap water, seeds were germinated in a continuous dark incubator at 25°C using the sand bed tissue culture method.After germination, seeds were irrigated with 1/4 nutrient solution.The formula of nutrient solution is shown in Table S1.The nutrient solution gradually increased from 1/2 concentration to total nutrient solution.During the culture period, pH of nutrient solution was adjusted to about 6.0.

Pb was added in the form of Pb(CH3COO)2·3H2O at concentrations of 0 (Pb0), 800 (Pb1), and 1600 (Pb2) mg/kg.N fertilizer was added in the form of NaNO3in the following concentrations:0.0 (N0), 0.1 (N1), 0.2 (N2), and 0.3 g/kg (N3) (Table 2).A total of 12 treatments were set for the whole experiment (Table 2), and each treatment was repeated three times.Phosphate and potash fertilizer were added in the form of KH2(PO4)3, and P2O5and K2O concentrations were 0.20 and 0.25 g/kg, respectively.The soil was mixed with Pb and base fertilizer, and loaded into a plastic pot (12.0 cm in diameter and 13.5 cm in height), to a bulk density of 1.4 g/cm3.After culturing inthe greenhouse for 2 weeks, seedlings with the same growth trend and height of 5 cm were transplanted to a pot with six seedlings for 8 weeks and watered appropriately to maintain 70% of the field capacity.

Table2 Experimental treatment

2.3 Sample collection

After 45 d of treatment, plants were harvested from roots and shoots, and potted soil samples were collected at the same time.Plant samples were treated at 105°C for 15 min, dried at 75°C to constant weight, and weighed.Samples were ground and sieved to determine the contents of nutrient elements and Pb.Soil samples were ground and sieved after air drying to determine soil pH, soil nutrient, and soil Pb contents.

2.4 Measurements

The forms of Pb in the soil were determined using the modified BCR (Bureau of Reference,European Community) three-step sequential extraction procedure (Rauret et al., 1999; Bao, 2000)(Table 3).Pb content of plants was determined by HNO3-HClO4digestion and inductively coupled plasma atomic emission spectrometry (Perkin Elmer, Waltham, USA).Total N,phosphorus (P), and potassium (K) contents of plants were determined using the Kjeldahl method.P was determined by the vanadium molybdenum yellow colorimetry method.K was determined by the flame photometer method after dry plant samples were digested by H2SO4-H2O2(Stanis?aw et al., 2017).The biomass was determined by drying the plants and weighing them with a 1/10,000 balance.Transfer coefficient of Pb is expressed as:Transport coefficient=aboveground Pb content/belowground Pb content.(1)

Table3 BCR (Bureau of Reference, European Community) three-step sequential extraction procedure

2.5 Statistical analysis

SPSS software package v.24.0 was used for data processing, and Origin Pro v.8.5 was used for visualizing the results.The variance method was adopted to analyze the significance of the relation between different amounts of-N and the adsorption of Pb by I.lactea (F test with a significance threshold of 0.05).Difference between treatments was tested by the LSD (least significant difference) method with a significance level of 0.05.

3 Results

3.1 Biomass

Under Pb0treatment, shoot biomass of I.lactea showed an overall decreasing trend with increasing-N concentration.Shoot biomass of I.lactea was not significantly different among CK, N2, and N3treatments, but significantly lower than N1treatment (Fig.1).Shoot biomass of I.lactea under Pb1treatment was slightly higher than those under Pb0and Pb2treatments, and root biomass decreased with the increase of-N concentration.Shoot biomass of I.lactea decreased significantly under Pb2treatment, and root biomass had no significant difference at N0and N1concentrations, but was substantially higher than those at N2and N3concentrations.Root/shoot ratio of I.lactea increased with the increase of-N concentration under Pb0and Pb2treatments, but decreased first and then slowly increased under Pb1treatment (Table S1).

Fig.1 Biomass of Iris lactea in lead (Pb)-contaminated soil with exogenous nitrate nitrogen (-N).CK, 0.0 g/kg Pb and N; N0, 0.0 g/kg; N1, 0.1 g/kg, N2, 0.2 g/kg; N3, 0.3 g/kg; Pb0, 0 mg/kg; Pb1, 800 mg/kg; Pb2, 1600 mg/kg.(a), Pb0 treatment; (b), Pb1 treatment; (c), Pb2 treatment.The detailed treatment of Pb and N is shown in Table 2.Different lowercase letters within the same Pb treatment indicate significant differences among different N treatments in root or shoot at P＜0.05 level according to Tukey's test.Bars are standard errors.

3.2 Effect of exogenous NO-3-N on soil pH

Soil pH generally showed a decreasing trend with increasing exogenous-N concentration under different Pb treatments (Fig.2).However, there were variations in pH in the soils of different Pb treatments.Under Pb0treatment, the addition of-N at all concentrations reduced soil pH to the same extent.Under Pb1treatment, exogenous-N at N2and N3concentrations reduced soil pH to the same extent.While under Pb2treatment,-N at N1concentration did not significantly affect soil pH, while-N at N2and N3concentrations similarly reduced soil pH.

Fig.2 pH value varied in Pb-contaminated soil with exogenous nitrate nitrogen (-N).CK, 0.0 g/kg Pb and N;N0, 0.0 mg/kg; N1, 0.1 g/kg, N2, 0.2 g/kg; N3, 0.3 g/kg; Pb0, 0 mg/kg; Pb1, 800 mg/kg; Pb2, 1600 mg/kg.The detailed treatment is shown in Table 2.Different lowercase letters within the same Pb treatment denote significant differences among different N treatments at P＜0.05 level according to Tukey's test.Bars are standard errors.

3.3 Uptake and transport of N by I.lactea

3.4 Chemical form distribution of Pb in the soil

In each treatment, the forms of Pb mainly exist in reducible state and residue state, accounting for more than 40% and 30% of the total Pb, respectively (Fig.3).Order of the proportion of different forms of Pb from high to low is reducible＞residual＞acid soluble＞oxidizable.With the increase in exogenous Pb, proportion of acid soluble Pb increased, while proportion of reducible and oxidizable Pb decreased, and residual Pb did not show an obvious change trend.Under Pb1treatment, addition of exogenous-N had no significant effect on the proportions of acid soluble, reducible, and residual Pb.However, proportion of oxidizable Pb significantly increased under Pb1N2treatment.

Fig.3 Transformation of lead (Pb) and proportions in different forms with exogenous nitrate nitrogen -N).N0, 0.0 mg/kg; N1, 0.1 g/kg, N2, 0.2 g/kg; N3, 0.3 g/kg; Pb1, 800 mg/kg; Pb2, 1600 mg/kg.(a), Pb1 treatment; (b),Pb2 treatment.The detailed treatment is shown in Table 2.Different lowercase letters within the same Pb treatment denote significant differences among different forms of Pb at P＜0.05 level according to Tukey's test.Bars are standard errors.

3.5 Absorption and transport of Pb by I.lactea

Under the treatments of Pb1and Pb2, root Pb concentration of I.lactea reached up to 1744.09 and 3893.24 mg/kg, respectively, while shoot Pb concentration reached up to 111.92 and 284.30 mg/kg, respectively.Uptake and accumulation of Pb in I.lactea were mainly concentrated in the root, and its root Pb content was 9.6-22.1 times higher than that of Pb content in the shoot (Fig.4).

Fig.4 Lead (Pb) content of Iris lactea in Pb-contaminated soil with exogenous nitrate nitrogen (-N).N0, 0.0 mg/kg; N1, 0.1 g/kg, N2, 0.2 g/kg; N3, 0.3 g/kg; Pb1, 800 mg/kg; Pb2, 1600 mg/kg.(a), Pb1 treatment; (b), Pb2 treatment.The detailed treatment is shown in Table 2.Different lowercase letters within the same Pb treatment denote significant differences among different N treatments at P＜0.05 level according to Tukey's test.Bars are standard errors.

Under Pb1treatment, with the increase in-N concentration, Pb concentration in the root of I.lactea increased first and then decreased (Fig.5a).Under Pb1N1treatment, Pb concentration reached the maximum, with 1744.09 mg/kg and the highest accumulation of 0.06 mg/plant, which was drastically higher than other treatments.Transport coefficients of Pb1N0＞Pb1N2＞Pb1N3＞Pb1N1in descending order were all less than 0.1 (Table S5).

Under Pb2treatment, addition of exogenous-N promoted the absorption of Pb in the root of I.lactea (Fig.5b).Addition of exogenous-N had no significant effect on Pb concentration in the shoot of I.lactea.But with the increase in-N concentration, transport coefficient decreased linearly, forming a descending order of Pb2N0＞Pb2N1＞Pb2N2＞Pb2N3.

Fig.5 Lead (Pb) accumulation content of Iris lactea in Pb-contaminated soil with exogenous nitrate nitrogen(-N).N0, 0.0 mg/kg; N1, 0.1 g/kg, N2, 0.2 g/kg; N3, 0.3 g/kg; Pb1, 800 mg/kg; Pb2, 1600 mg/kg.(a), Pb1 treatment;(b) Pb2 treatment.The detailed treatment is shown in Table 2.Different lowercase letters within the same Pb treatment denote significant differences among different N treatments at P＜0.05 level according to Tukey's test.Bars are standard errors.

4 Discussion

4.1 Variation of nitrogen on soil pH

The selection of appropriate N fertilizer forms and fertilization methods can be used as a strategy to control the absorption of heavy metals by crops and improve the safety of agricultural products(Hamlin and Barker, 2006).The effects of chemical N fertilizer on soil physical-chemical properties are mainly manifested via the effect on soil acidity.The reasons for change in soil pH can be attributed to two aspects.Firstly, pH change is caused by the transformation and migration of N form.When-N is applied to the soil, soil pH can be reduced significantly over a relatively short period through nitrification, and then-N produced in the nitrification process or applied in the form of fertilizer can take away the base ions through leaching, further acidifying the soil (Williams et al., 1987).Secondly, change in rhizosphere pH caused by plant absorption of different forms of N and secretion of H+or OH-.When different forms of N fertilizer are applied to plants, the plant rhizosphere environment exhibits distinct changes, with the root system secreting different ions after-N and-N uptake.Following-N uptake,a root system will secrete H+, which causes acidification around the rhizosphere, while following-N uptake, the root system secretes OH-, resulting in rhizosphere alkalization (Wallace et al.,1979).Different forms of N fertilizer have different effects on soil acidification and rhizosphere environment, thus resulting in the variation of adsorption of heavy metals in the soils (Lou et al.,2005).

4.2 Effect of nitrate nitrogen addition on lead content

Previous studies have reported different forms of Pb in the soil (Wu et al., 2013).Rosik-Dulewska and Karwaczyńska (2004) found that Pb enters the soil mainly in the form of residue; conversely,Lee et al.(2015) showed that Pb was predominantly found in carbonate and reducible fractions in soil.In this study, we found that Pb mainly exists in a reducible state after entering the soil but that residual state also accounts for a large proportion.Pb content in the reducible and residual states accounts for ＞78% of the total amount.The reason for the differences among these studies may be related to the soil used.There is a very significant and positive correlation between Pb contents of root and shoot of I.lactea and Pb contents of various forms in the soil, among which the correlation with the content of acid soluble Pb is the most significant (Table S6).The stepwise regression analysis was made between the lead content in root and shoot of I.lactea and the acid soluble Pb content in soil.The regression equation was as follows: Y1= -222.58+18.99X,R2=0.944, P＜0.05; Y2= -3.85+1.24X, R2=0.810, P＜0.05, where Y1 and Y2 is the lead content(mg/kg) of shoot and root, respectively; X is the acid soluble lead content (mg/kg).The results confirmed that Pb absorbed by the root and shoot of I.lactea changed into acid soluble Pb in the soil.The addition of exogenous-N had a positive impact on the form of Pb in the soil.

4.3 Effect of nitrate nitrogen addition on absorption and transport of lead

The low Pb content in the shoot of I.lactea will not stress the normal growth of I.lactea, but the amount of Pb it can absorb is far beyond the tolerance of most plants to Pb in nature (Yuan et al.,2015).Moreover, the addition of Pb did not affect the content and distribution proportion of various forms of Pb in the soil (Wang et al., 2018).Therefore, the direct effect of-N may be the reason for the increase of Pb absorption in the root of I.lactea.It is presumed that the increase in-N concentration brings more anion charge in the soil, prompting more anion uptake by the plant, which requires more cation uptake by the plant for balance, facilitating the membrane transport process of cations.Therefore, Pb content absorbed by the plant root increases.Moreover,the addition of exogenous-N promotes the synthesis of organic acids in the soil.Organic acids may combine with Pb and assist in Pb translocation and accumulation (Monsant et al., 2010).Studies have proven that most of-N absorbed with the aid of plant root desires to be reduced and utilized by plant, while a small part is stored in plant vacuoles as substances for ion balance and osmotic regulation.When-N in the soil exceeds the upper limit of nitrogen required by plants in growth period, it may cause ion imbalance in plants.Moreover, excessive nitrogen will also inhibit the absorption of other essential elements by plants, thus affecting the growth of plants (Kastl et al., 2015).In this experiment, under the condition of Pb2treatment, the addition of-N substantially inhibited the normal growth of the aboveground part of I.lactea, but had a relatively small impact on the root.This is partly due to the stress effect of the rise of Pb concentration on the plant, but more is the inhibitory effect of the addition of exogenous-N on the growth of I.lactea.

4.4 Relationship between nutrient absorption and transport of lead

Shoot N content of I.lactea negatively correlated with shoot P content, shoot and root Pb contents,and four forms of Pb (Fig.6).Shoot N content of I.lactea positively correlated with root P content and shoot and root dry weights.There was a correlation between root N accumulation and shoot dry weight (Fig.6).It can be noted that with the addition of NO-3-N, the accumulation of P in root increased considerably, and most P element remained in plant root.N element is transported to the aboveground part of plant in huge quantities and contributes to the dry weight of plant (Xu et al., 2019).A small amount of N promotes plant dry weight and the absorption of Pb.With the increase of-N concentration, the absorption capability of I.lactea to Pb decreases.

Fig.6 Correlations of Iris lactea growth with absorption and transport of Pb.*, P＜0.05 level.

5 Conclusions

The present study was conducted to study the effects of exogenous addition of-N on the uptake and transport of Pb and nutrients, as well as the growth of I.lactea.The results found that exogenous-N mainly affected the content of Pb in the root of I.lactea.Under different Pb concentrations,-N application inhibited the transport of Pb from root to shoot.The higher the Pb concentration, the more significant the effect.This may be due to the balance effect of ions absorbed by the root of I.lactea caused by the addition of-N.In the case of a certain degree of soil Pb pollution, the migration of heavy metals in plants can be improved by adjusting the level of soil nutrition.The addition of exogenous-N reduces soil pH and affects the distribution and content composition of different forms of Pb in the soil to a certain extent.This is due to the combination ofand base ions leads to the reduction of soil pH underneath the motion of leaching.The demand for-N could be very low in I.lactea.In the soil polluted by different concentrations of Pb, the addition of exogenous-N can also promote the growth of I.lactea, but there is a limit in the concentration range of exogenous-N.When the concentration of-N is 0.1 g/kg, the promotion effect is the best.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (51978659).