Polyaspartic acid mediates the absorption and translocation of mineral elements in tomato seedlings under combined copper and cadmium stress

HU Mei-mei, DOU Qiao-hui, CUl Xiu-min, LOU Yan-hong, ZHUGE Yu-ping

National Engineering Laboratory for Effcient Utilization of Soil and Fertilizer Resources/College of Resource and Environment, Shandong Agricultural University, Tai'an 271018, P.R.China

Abstract Polyaspartic acid (PASP) is a nontoxic, biodegradable, environmentally friendly polymer and is widely used as a fertilizer synergist in agricultural production. In many old orchards and vegetable gardens, highly fertile soil is often accompanied by severe heavy metal contamination. The present study was designed to investigate mineral element interactions mediated by PASP under copper (Cu)+cadmium (Cd) combined stress to provide reasonable suggestions for scientif ic fertilization. A pot experiment was conducted in which tomato seedlings served as plant materials. A concentration of 700 mg L-1 PASP and foliar spraying application methods were selected based on previous experiments. Four treatments were applied: normal soil (control (CK)), Cu+Cd (combined stress), Cu+Cd+PASP, and normal soil+PASP. The plant biomass, root activity, and mineral elements were measured, and these data were analyzed by Data Processing System (DPS) statistical software. The results showed that, under Cu+Cd combined stress, PASP promoted stem diameter growth, root activity and chlorophyll content and ultimately increased the biomass of tomato seedlings to different degrees. Moreover, the content of both Cu and Cd and their individual accumulation in plants decreased. PASP increased the distribution of Cu and Cd in the roots under Cu+Cd combined stress, and the translocation ability from the roots to shoots was signif icantly restricted. With respect to essential elements, PASP promoted mainly the absorption and translocation of potassium (K), calcium (Ca), and magnesium (Mg), which greatly exerted physiological roles. However, the variation trends of Cu and Cd under normal soil conditions contrasted with those under stress conditions. With respect to essential elements other than K, Ca, and Mg, PASP mostly restrained their absorption but promoted their translocation. The regulatory mechanism of PASP differed between the combined stress conditions and normal soil conditions. Under the combined stress conditions, PASP seemed to mainly promote these advantageous factors that exert physiological regulatory functions. Under normal soil conditions, PASP mainly acted as a biological stimulant or signaling molecule for increased nutrient eff iciency, which caused greater biomass productivity.

Keywords: tomato seedlings, polyaspartic acid, Cu+Cd combined stress

1. lntroduction

Heavy metals are prevalent in modern cities due to rapid urbanization and industrial development (Liu et al. 2016; Pan 2018). In recent years, due to the unnecessary applications of pesticides and organic and chemical fertilizers as well as industrial waste, the content of heavy metals in agricultural soils has risen. The main pollutants are cadmium (Cd), nickel (Ni), copper (Cu), arsenic (As), mercury (Hg), lead (Pb) and so on (EPMLR 2014). As an essential trace element, Cu is also a heavy metal that is toxic to plants when absorbed in excess. Cd is a wellknown and widespread heavy metal (Xu et al. 2010) and is considered the most toxic pollutant in the soil (Satarug et al. 2003; Wei and Zhou 2006). Although Cu and Cd are not typically associated metals, in the soil, they often exist synchronously in the form of a compound pollutant (Wang et al. 2016). Cu and Cd in agricultural soils can enter into plants, ultimately leading to severe threats to human health via food consumption. Therefore, establishment of remediation programs to reduce the risk through the soil-food-human pathway is urgent.

Polyaspartic acid (PASP) is a type of fertilizer synergist with carboxylic acid side chains; PASP is completely biodegradable and environmentally friendly. Previous studies have shown that PASP or PASP-urea can enhance plant nutrient-absorbing ability as well as the growth rate, biomass, grain yield and quality when applied to corn, rice, mustard green and other plants (Koskan et al. 1999; King et al. 2012; Deng et al. 2015). Moreover, as an extraction agent, PASP could effectively separate the Pb2+and Cd2+in sludge, and the exchange rate reached 90% (Zhu et al. 2005). Thus, PASP can promote plant growth directly or indirectly by chelating heavy metal ions to reduce biological toxicity. However, the effects of PASP can differ depending on the fertility status of the soil. Therefore, this experiment aimed to investigate the responsive regularity of PASP applied to tomato seedlings under Cu+Cd combined stress and normal soil conditions to provide theoretical evidence for fertilizing on contaminated soil.

2. Materials and methods

2.1. Experimental description

Tomato (Solanum lycopersicum L.) variety Zhongshu 4 was used in this study. The tested soils were collected from the surface layer (0-20 cm) of the test area. The soil type was brown earth, whose basic properties are shown in Table 1.

A pot cultivation experiment was conducted in a greenhouse (natural light; 25 to 32°C during the day and 19 to 26°C during the night; 75% relative humidity). Four different treatments were applied: (1) control (CK); (2) Cu+Cd; (3) Cu+Cd+PASP; and (4) PASP. Exogenous Cu and Cd were applied evenly to the soil as solutions in the form of CuCl2·2H2O and Cd Cl2·2.5H2O; the concentrations were 500 and 8 mg kg-1, respectively. After they were polluted, the soils were air-dried, mixed thoroughly, sifted through a 40-mesh screen and then added to plastic pots (4.0 kg pot-1). The relative water content was maintained at 80% and selfbalanced for 3 wk. Before f ield planting, the available Cu and Cd were 264.09 and 7.55 mg kg-1, respectively. On March 10, tomato seeds were sown into plug trays. On April 8, at which point the tomato seedlings had developed f ive or six true leaves, the seedlings displaying uniform growth vigor were selected for transplantation to plastic pots (Φmax=22.5 cm, Φmin=16.3 cm, H=19.5 cm); one seedling was transplanted per pot, and each treatment consisted of eight pots. When the seedlings developed seven or eight true leaves, the plants were treated with PASP.

Previous experiments indicated that plants grew better when their leaves were treated with PASP sprays than when their roots were treated with PASP soil applications. A practical concentration of PASP was 700 mg L-1. The seedlings were sprayed with 100 mL pot-1PASP once every other day; the CK seedlings were sprayed with water. All pots were arranged randomly. After 20 days, the seedlings were harvested and washed thoroughly with running tap water followed by distilled water for 3 min. The biomass (fresh weight (FW)) was measured directly with an electronic balance and then heated to 105°C for 30 min to terminate enzyme activity, after which the biomass was subjected to constant drying at 70°C until the weight became constant. Afterward, the biomass was milled, sieved, sealed and preserved.

Table 1 The physical and chemical properties of the potted soil

2.2. Determination of the physical and chemical properties of soil

The p H, electrical conductivity, available nitrogen (N), available phosphorus (P), and available potassium (K) were analyzed in accordance with previous methods (Bao 2002).

2.3. Determination of the root activity of and chlorophyll content in plants

The root ac tivity was d etermined by the 2,3,5- triphenyltetrazolium chloride (TTC) dyeing method (Wang et al. 2016), and the chlorophyll content was determined by the 80% acetone extraction method.

2.4. Determination of Cu and Cd content in plants

Plant samples were digested in HNO3-HClO4(4:1, v/v), and the f iltrate was analyzed to determine the content of Cu and Cd by an atomic absorption spectrophotometer (Shimadzu Corporation AA7000).

2.5. Analysis of N, P and K

Some plant samples were digested with H2SO4-H2O2. The N content was determined by Kjeldahl azotometer, the P content was determined by the color comparison method with ammonium vanadate-molybdate, and the K content was determined by f lame photometry.

2.6. Analysis of Ca, Mg, Fe, Zn, and manganese

Some plant samples were digested with HNO3-HClO4(4:1, v/v), and the f iltrates were diluted at various times to determine the content of Ca, Mg, Fe, Zn, Mn via an atomic absorption spectrophotometer (Shimadzu AA-7000) (Lu 2000).

2.7. Statistical analysis

Microsoft Excel software was used for data processing and f iguring, and the least signif icant difference (LSD) was used for multiple comparisons between different treatment means.

3. Results

3.1. Growth potential of tomato seedlings under different Cu and Cd treatments

Fig. 1-A and B show that the effects of different Cu, Cd, and PASP treatments on tomato growth were essentially similar. The plant height and stem diameter of tomato were clearly restricted by Cu+Cd combined stress, and exogenous PASP could not effectively ameliorate the negative effects. Spraying PASP alone had no obvious effects on plant height but notably promoted stem diameter growth, which was benef icial to the development of robust seedlings.

Fig. 1 The growth vigor of tomato seedlings under different physiological conditions. FW, fresh weight. CK, control; PASP, polyaspartic acid. Different letters indicate signif icant differences at the 5% level. The error bars indicate SE (n=3).

Compared with that of the CK seedlings, the root biomass of the seedlings subjected to Cu+Cd combined stress signif icantly decreased (Fig. 1-C). However, spraying PASP signif icantly alleviated the root growth inhibition. A similar trend was observed in the aboveground biomass. Under normal soil conditions, 700 mg L-1PASP signif icantly promoted both the root and aboveground growth of tomato plants. This f inding indicated that PASP could somewhat alleviate the damage caused by heavy metals (Cu+Cd) and that the enhancement effect was more apparent under normal growth conditions.

3.2. Root activity of and chlorophyll content in tomato seedlings under different conditions

As shown in Fig. 2-A, compared with that of the CK seedlings, the root activity of tomato seedlings treated with Cu+Cd combined stress markedly decreased by 26.94%. However, the inhibitory effect diminished after the spraying of exogenous PASP, and the root activity recovered to nearly the same level as that of the CK seedlings. Under normal growth conditions, spraying PASP signif icantly increased the root activity (57.02% higher activity than that of the CK seedlings), which indicated that PASP had a strong effect on restoring the root function of tomato seedlings.

As shown in Fig. 2-B, compared with the CK treatment, the Cu+Cd treatment caused a downward trend in chlorophyll content. Although spraying PASP could clearly counteract the decline, the chlorophyll content still did not return to the level of chlorophyll content in the CK treatment under combined stress. However, little difference was found in chlorophyll content between the PASP treatment and CK treatment. Thus, PASP might exert regulatory functions, especially concerning the synthesis of chlorophyll under Cu+Cd combined stress.

3.3. Absorption and distribution of Cu in tomato seedlings under different Cu and Cd treatments

Table 2 shows the variation trend of Cu under different treatments. Under Cu+Cd combined stress, the Cu content in each organ markedly increased. In the roots, Cu content sharply increased to 2.91 times that of the CK. The distribution ratio showed that the excessive amount of Cu which was passively absorbed was mainly immobilized in the roots. The addition of 700 mg L-1PASP clearly relieved the trend of increasing Cu content and inhibited excessive Cu translocation to the leaves.

The absorption and translocation of Cu each exhibited vastly different trends in response to PASP treatment. Under normal growth conditions, applying PASP signif icantly restricted roots from absorbing Cu from the media (only 23.45% of that of the CK) but promoted limited Cu translocation from the roots to aboveground parts, which caused 60.06% of Cu to be distributed in the leaves. The regulatory mechanism of PASP therefore clearly differed between the combined stress and normal conditions.

3.4. Absorption and distribution of Cd in tomato seedlings under different treatments

Fig. 2 The root activity of and chlorophyll content in tomato seedlings under different treatments. CK, control; PASP, polyaspartic acid. Different letters mean signif icant differences at the 5% level. The error bars indicate SE (n=3).

Table 2 The accumulation and distribution of copper in tomato plants

Table 3 The accumulation and distribution of cadmium in tomato seedlings

Compared to the CK treatment, under the combined stress treatment, the Cd content markedly increased, up to 35.8, 63.6 and 28.8 times greater in the roots, shoots and leaves, respectively. To some extent, adding PASP could restrict the absorption and translocation of Cd but could not reverse the trend of accumulation. The excessive amounts of Cd passively absorbed tended to be stored in the roots and shoots. Under normal growth conditions, spraying PASP could strongly reduce the absorption and accumulation of Cd. Furthermore, 69.81% of the limited Cd was distributed in the leaves. This phenomenon was similar for Cu, despite the translocation eff iciency of Cd being much higher than that of Cu.

3.5. Absorption and distribution of N, P, and K in tomato seedlings under different Cu and Cd treatments

As shown in Fig. 3-A, compared to the CK treatment, the Cu+Cd combined stress treatment promoted root absorption of N from the media and enhanced the translocation of N from the roots to leaves. However, adding PASP exhibited no clear effects. Under normal growth conditions, adding PASP evidently reduced the absorption and translocation of N, especially in the leaves, in which the N accumulation decreased by 9.43%.

As shown in Fig. 3-B, the absorption and translocation of P were restricted by Cu+Cd combined stress, and exogenous PASP could effectively relieve this restriction in the roots; however, in the leaves, the decrease was enhanced. Applying PASP separately signif icantly reduced the P content in the roots and leaves.

As shown in Fig. 3-C, the K content was noticeably restricted in the leaves by only Cu+Cd combined stress, and exogenous PASP effectively relieved the adverse effects in the leaves and stems. However, under normal soil conditions, spraying PASP resulted in a progressive trend in the absorption and translocation of K, but this trend was signif icant only in the roots and stems.

3.6. Absorption of other similarly charged elements in tomato seedlings under different Cu and Cd treatments

Fig. 3 The content and accumulation of N, P, and K in tomato plants under different Cu and Cd treatments. CK, control; PASP, polyaspartic acid. Different letters mean signif icant differences at the 5% level. The error bars indicate SE (n=3).

As shown in Table 4, compared to the CK treatment, the Cu+Cd combined stress treatment markedly inhibited the translocation of Fe from the roots to the leaves, which led to 15.07 and 34.83% reductions in Fe content in the stems and leaves, respectively. PASP foliar sprays effectively promoted the translocation of stored Fe from the roots to leaves, which resulted in increased Fe contents of 165.12 and 31.4% in the stems and leaves under combined stress, respectively. However, spraying PASP separately caused all Fe contents to decrease (21.4, 11.42, and 31.96% in the roots, stems, and leaves, respectively).

The variation trend of Mn was similar to that of Fe. The Mn content decreased by 36.59, 33.51, and 27.47% in the roots, stems and leaves, respectively; these decreases were greater than those of the Fe content under Cu+Cd combined stress, and the repairing effect of PASP was ref lected mainly in the stems (40.86%). Under normal growth conditions, PASP signif icantly reduced the absorption and translocation of Mn, especially in the roots, in which the Mn content decreased by 34.22%.

Compared with the CK treatment, the combined stress treatment signif icantly reduced the absorption and translocation of Zn. Exogenous PASP could eff iciently alleviate the inhibition effect but could not restore the Zn content to that in the CK treatment. Under normal soil conditions, spraying PASP also caused the Zn content to clearly decrease by 37.01 and 62.30% in the roots and leaves, respectively.

A summary of the above analysis shows that, under excessive Cu+Cd stress, the essential elements that have a similar charge as that of both Cu and Cd were apparently competitively inhibited; to some degree, exogenous PASP could alleviate this inhibition effect. Under normal conditions, without exception, exogenous PASP reduced the absorption of these nutrient elements.

However, the combined Cu+Cd stress promoted the absorption and translocation of Ca by up to 24.95, 51.9, and 25.2% in the roots, stems and leaves, respectively. In addition, adding PASP maintained the increasing trend. Under normal conditions, exogenous PASP inhibited the absorption and translocation of Ca by 12.99, 18.76, and 16.36% in the roots, stems and leaves, respectively. The variation trend of Mg was similar to that of Ca under both the combined stress and normal conditions.

4. Discussion

PASP, which contains a large number of carboxylic groups and amide groups among its molecular chains (Tomida et al. 1997), strongly absorbs ions that reduce nutrient loss and improved nutrient accumulation in the soil (Kinnersley et al. 1997). Therefore, increased levels of available nutrients in the root feeding zone occur in response to PASP applications, and more eff icient nutrient use is achieved; in addition, the availability of toxic ions (such as Cr6+) can decrease (Koskan et al. 1999; Du et al. 2011; Fu et al. 2017). In this experiment, under Cu+Cd combined stress, spraying PASP on the leaves also strengthened the resistance and tolerance of tomato seedlings against heavy metal toxicity, clearly restoring plant biomass, especially that of the roots, much higher than that of the CK seedlings. However, for plant height and stem diameter, the relieving effect exhibited just a gradually increasing trend, which was likely because the treatment time was short. However, under normal conditions, foliar spraying of PASP increased dry matter accumulation. Although plant height changed little in response to PASP, root activity was strongly promoted, and stem diameter markedly increased. These results indicated that the roots could function with increased eff iciency to absorb and translocate mineral nutrients.

Table 4 The content and accumulation of other cations in tomato plants under different Cu and Cd treatments

Root activity was closely related to the whole growth environment; the root activity directly ref lected root absorption function but was easily negatively affected by heavy metal stress (Hawrylak-Nowak et al. 2015). In this study, regardless of the stress conditions, spraying PASP signif icantly promoted root activity. Moreover, in the aboveground parts, the suff icient amount of detected chlorophyll in the leaf conf irmed that the plants could function properly (Singh et al. 2007). Both of these aspects ensured that the tomato seedlings could perform normal metabolic activity, which led to the recovery of or the increase in root biomass.

The effects of PASP on other mineral elements varied. For Cu and Cd, under excessive stress, PASP played a regulatory role in preventing excessive toxic ions from being absorbed and translocated mineral nutrients to the leaves, which ensured normal cell protoplasm function as much as possible. However, under normal soil conditions, PASP sharply prevented mineral absorption, but to a greater extent, PASP limited the translocation of Cu and Cd to the leaves. This phenomenon was inconsistent with previous research results (Leng 2005). Here, the results seem to imply that PASP actually functions somewhat as a biological stimulant under nonstress conditions (Pamela Calvo et al. 2014).

Cu+Cd combined stress clearly restricted the absorption of the essential elements N, P, Fe, Mn, and Zn, while spraying PASP effectively alleviated the inhibition and positively regulated the translocation of these ions to the leaves. Under Cu+Cd combined stress, the absorption of Fe increased in response to PASP, which was consistent with the trend of chlorophyll content. Under normal soil conditions, spraying PASP exhibited a passive role in the absorption and translocation of some trace elements. Regardless of the presence of stress, PASP always positively affected the absorption and translocation of only K+. This result indicates that PASP exhibits regulatory ability by activating the physiological mechanism of K+(Marschner 2013). Unintentionally combined stress promoted the absorption and translocation of Ca and Mg, and PASP intensif ied this trend. This result was likely due to the special relation between Ca and PASP. As a green scale inhibitor or desalinator (Shilpa et al. 2014), the carboxylic groups among the PASP polymer are the main functional groups that inhibit the formation of calcium carbonate and calcium sulfate scale, while the hydroxylic groups and acylamino groups are the main functional groups that inhibit the formation of calcium phosphate (Chen et al. 2015). These results provided a fundamental understanding of why suff icient amounts of active Ca are essential for growth under abiotic stress (Marschner 2013). Under normal conditions, PASP clearly reduced the absorption and translocation of Ca and Mg.

Therefore, from a general perspective of the above elements, the variation among the essential elements was extremely large. When exposed to compound heavy metal stress, some elements are competitively inhibited due to their similar chemical characteristics (Tanhan et al. 2007; Hawrylak-Nowak et al. 2015). Some elements, especially essential key elements (such as N and P), were strongly limited. PASP could somewhat relieve the negative effects but barely restored the contents to those in the CK treatment. When exposed to combined stress, K, Ca and Mg exhibited more physiological function and osmotic adjustment, which helped maintain the integrity of the structure and function of the cell (Marschner 2013).

It is well known that PASP can chelate Ca, Mg, Cu, and Fe as well as other polyvalent metal ions (Koskan and Low 1992). The functional groups of the amino acids on the end and the carboxylic acids on the side chains can coordinate with metal cations and encapsulate them into a “hole”, which disperses and stabilizes metal cations in solution (Gutierrez et al. 1999). However, spraying PASP alone on the leaf surface reduced the absorption of some mineral elements under normal soil conditions, but root activity, stem diameter and plant biomass were instead promoted; these indexes are key indicators of seedling quality. Furthermore, the tomato seedlings showed no obvious nutrient def iciency symptoms, which meant that their nutrient use eff iciency increased. This phenomenon is inconsistent with previous reports on PASP as a fertilizer synergist (Du et al. 2011; Deng et al. 2014, 2015). In addition, the response mechanism distinctly differed between PASP foliar sprays and root applications.

5. Conclusion

The above evidence suggests that, in a healthy soil environment, PASP plays a role similar to a biological stimulant or signaling molecule to regulate the absorption and translocation of mineral nutrients, whereas under stress conditions, PASP acts more like a fertilizer synergist. However, additional studies are needed to elucidate the response mechanism.

Acknowledgements

This work was supported by the Project of Shandong Province Higher Educational Science and Technology Program, China (J16LF02), the Funds of Shandong “Double Tops” Program, China (SYL2017YSTD01), and the Major Scientif ic and Technological Innovation Project in Shandong Province, China (2018CXGC0209).