Humic + Fulvic acid mitigated Cd adverse effects on plant growth, physiology and biochemical properties of garden cress

Cadmium (Cd) is a toxic and very mobile heavy metal that can be adsorbed and uptaken by plants in large quantities without any visible sign. Therefore, stabilization of Cd before uptake is crucial to the conservation of biodiversity and food safety. Owing to the high number of carboxyl and phenolic hydroxyl groups in their structure, humic substances form strong bonds with heavy metals which makes them perfect stabilizing agents. The aim of this study was to determine the effects of humic and fulvic acid (HA + FA) levels (0, 3500, 5250, and 7000 mg/L) on alleviation of Cadmium (Cd) toxicity in garden cress (Lepidium sativum) contaminated with Cd (CdSO4.8H2O) (0, 100, and 200 Cd mg/kg) under greenhouse conditions. Our results showed that, Cd stress had a negative effect on the growth of garden cress, decreased leaf fresh, leaf dry, root fresh and root dry weights, leaf relative water content (LRWC), and mineral content except for Cd, and increased the membrane permeability (MP) and enzyme (CAT, SOD and POD) activity. However, the HA + FA applications decreased the adverse effects of the Cd pollution. At 200 mg/kg Cd pollution, HA + FA application at a concentration of 7000 mg/L increased the leaf fresh, leaf dry, root fresh, root dry weights, stem diameter, leaf area, chlorophyll reading value (CRV), MP, and LRWC values by 262%, 137%, 550%,133%, 92%, 104%, 34%, 537%, and 32% respectively, compared to the control. Although the highest H2O2, MDA, proline and sucrose values were obtained at 200 mg/L Cd pollution, HA + FA application at a concentration of 7000 mg/L successfully alleviated the deleterious effects of Cd stress by decreasing H2O2, MDA, proline, and sucrose values by 66%, 68%, 70%, and 56%, respectively at 200 mg/kg Cd pollution level. HA + FA application at a concentration of 7000 mg/L successfully mitigated the negative impacts of Cd pollution by enhanced N, P, K, Ca, Mg, Fe, Mn, Cu, Mn, Zn, and B by 75%, 23%, 84%, 87%, 40%, 85%, 143%, 1%, 65%, and 115%, respectively. In addition, HA + FA application at a concentration of 7000 mg/L successfully reduced Cd uptake by 95% and Cl uptake by 80%. Considering the plant growth parameters, the best results were determined when HA + FA concentration was 7000 mg/L. We have shown that, it is critical to apply a humic substance with high percentage of FA, which was 10% in this study, to mitigate the adverse effects of heavy metal stress on plant growth. In conclusion, the application of HA + FA may be suggested as an effective solution for reducing the Cd uptake of the plants by stabilizing Cd in soil and preventing translocation of Cd from the roots of plant to its shoot and leaves.


Materials and methods
Setting up the experiment. Garden cress (Lepidium sativum cv Helen) was grown in polyethylene pots in the greenhouse of Atatürk University, Erzurum. The greenhouse temperature was maintained at an average temperature of 22 (± 2)°C during the day, and 17 (± 2)°C at night.
For heavy metal stress treatments, cadmium (CdSO 4 .8H 2 O) was mixed with the medium at three different concentrations (0, 100, and 200 mg/kg) and incubated for 3 weeks.
To prepare HA + FA (Powhumus) solutions 350 g Powhumus was dissolved in 1 L water (350 000 mg/L) and then diluted to obtain three different concentrations (3500, 5250, and 7000 mg/L). HA + FA solutions were applied to the soil three times a week, starting the day before planting. As a control, 0 mg/L HA + FA was used, instead 150:100:150 kg/ha NPK was mixed into the medium as basal fertilizer.
Seeds of garden cress were sown in pots filled with 1 L of three-week-incubated garden soil: sand (1:1, v:v) mixture in a 1-1.5 cm depth, with 10 seeds per pot. After the seedling emergence, four plants were left in each pot with the same appearance. Thiobarbituric acid-reactive substances were measured as MDA, a degraded product of the lipid, which determines the lipid peroxidation. The concentration of MDA was determined from the absorbance curve, by using an extinction coefficient of 155 mmol L −1 cm −1 .
Sucrose and proline analysis. Sucrose concentration was measured by a method given by Chopra et al. 14 .
Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activity. CAT, POD, and SOD activities were determined based on the method given by Sahin et al. 16 .
Mineral analysis. Garden cress leaves were ground after being dried at 68 °C for 48 h in an oven. Determination of the total N was achieved by the Kjeldahl method using a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Konigswinter, Germany). An inductively coupled plasma spectrophotometer (Optima 2100 DV, ICP/ OES; Perkin-Elmer, Shelton, CT) was used to determine tissue P, K, Ca, Mg, Fe, Cu, Mn, Zn, B, Cl and Cd 17,18 . Statistical analysis. In the experiment, a randomized plot design was used and the obtained data were analyzed using SPSS 20 statistical package program. Data were subjected to variance analysis (ANOVA) and differences of means were determined by Duncan multiple comparison test.

Results
As can be seen in Table 1, humic + fulvic acid application affected the leaf fresh, leaf dry, root fresh and root dry weights of garden cress significantly under Cd stress condition. Our results showed that Cd pollution negatively affected all parameters investigated and the negative effect increased with the increased pollution doses. Without Cd pollution, the highest leaf fresh and leaf dry weight of the plant were obtained when HA + FA was applied at a concentration of 7000 mg/L, whereas the highest root fresh and root dry weight were found when the concentration of HA + FA applied was 3500 mg/L. These findings indicate that, without Cd pollution, leaf fresh weight and dry weight increased 35% and 30%, respectively with application of 7000 mg/L HA + FA, whereas root fresh weight and dry weight increased 33% and 15% when 3500 mg/L HA + FA was applied, compared to the control (HA + FA = 0 mg/L). With Cd pollution, leaf fresh, leaf dry, root fresh and root dry weights were affected negatively. However, the HA + FA applications alleviated the deleterious effects of the Cd to the plant. At 200 mg/kg Cd pollution, HA + FA application at a concentration of 7000 mg/L increased the leaf fresh, leaf dry, root fresh and root dry weights by 262%, 137%, 550% and 133%, respectively, compared to the control ( Table 1).
The effect of HA + FA applications on the stem diameter, leaf area and CRV of the garden cress under Cd stress is presented in Table 2. Cd pollution significantly decreased the stem diameter, leaf area and CRV in the cress. The most negative effect occurred in 200 mg/kg Cd pollution. However, HA + FA applications reduced the negative effects of 100 and 200 mg/kg Cd stress on the cress. At 200 mg/kg Cd pollution, HA + FA application at a concentration of 7000 mg/L increased the stem diameter, leaf area and CRV by 92%, 104%, and 34%, respectively, compared to the control ( Table 2). Table 1. Effect of cadmium and HA applications on leaf fresh, leaf dry, root fresh and root dry weight of garden cress. The difference between the means indicated by different letters in the same column is statistically significant (Duncan multiple comparison test, P < 0.05). www.nature.com/scientificreports/ The present study showed that Cd pollution increased the MP value in the cress while decreasing the LRWC value. The highest MP and the lowest LRWC values were determined in plants treated with 200 mg/kg Cd. The HA + FA applications mitigated the adverse effects of Cd and at 200 mg/kg Cd pollution, HA + FA application at a concentration of 7000 mg/L increased the MP and LRWC values by 537% and 32%, respectively, compared to the control (Table 2).
CAT, POD, and SOD activities were found to increase with increasing Cd pollution. At 200 mg/kg Cd pollution, 7000 mg/L HA + FA application decreased CAT and SOD activities by 43% and 21% respectively, and increased POD activity by 186%, compared to the samples without HA + FA application (Table 3). Although the highest H 2 O 2 , MDA, proline and sucrose values were obtained at 200 mg/kg Cd pollution, HA + FA application at  (Table 3). Cd pollution decreased all mineral elements in garden cress investigated within this work, but for the cadmium itself. On the other hand, application of HA + FA, enhanced the mineral element content of the garden cress except for Cd (Tables 4, 5). The highest N, P, K, Ca, Mg, Fe, Mn, Cu, Mn, Zn, B, and Cl were obtained from 7000 mg/L HA + FA application without Cd pollution, while the lowest values were determined from 0 ml HA + FA with 200 mg/kg Cd pollution treatment. HA + FA application at a concentration of 7000 mg/L successfully alleviated the deleterious effects of Cd stress by increased N, P, K, Ca, Mg, Fe, Mn, Cu, Mn, Zn, and B by 75%, 23%, 84%, 87%, 40%, 85%, 143%, 1%, 65%, and 115%, respectively. On the other hand, HA + FA application Table 4. Effect of cadmium and HA applications on macro element content of garden cress. The difference between the means indicated by different letters in the same column is statistically significant (Duncan multiple comparison test, P < 0.05). www.nature.com/scientificreports/ at a concentration of 7000 mg/L successfully reduced Cd and Cl uptake, by 95% and 80%, respectively (Tables 4,  5).

Discussion
Heavy metals often cause toxic effects on plants such as chlorosis, inhibition of growth and photosynthesis, water balance variations and nutrient assimilation, which ultimately lead to plant death 19 . Heavy metals exhibit toxicity through four suggested mechanisms in plants. These are: (i) competition with nutrient cations for adsorption at the root surface (e.g. competition of As and Cd with P and Zn, respectively, for adsorption); (ii) inactivation of sulfhydryl groups (-SH) by direct interaction, which disrupts plant's structure and function; (iii) collapsing the function of enzymes by displacing essential cations from specific binding sites; and (iv) the production of active oxygen species (ROS), which ultimately damage macromolecules 20 . Cd is considered an important pollutant because of its high toxicity and high water solubility. Cadmium can alter mineral uptake through its effects on the presence of minerals in the soil or through a reduction in the population of soil microbes. Cadmium may have a negative effect on stomatal conductivity, perspiration, and photosynthesis efficiency. Chlorosis, leaf folds, and scrub are the common and easy-to-see symptoms of cadmium toxicity of plants. Cd also decrease nitrate uptake and its transport from the roots to the shoots by inhibiting nitrate reductase activity 21 .
In this study, Cd stress was found to affect the growth of garden cress negatively (Table 1). While the LRWC decreased in the cress grown under Cd stress, the MP increased (Table 2). Similarly, previous studies showed the negative effects of Cd stress on plant growth characteristics in radish 22 , garden cress 23,24 and lettuce 25 .
On the other hand, HA + FA applications in cress grown under Cd stress positively affected the plant growth (Table 1). Furthermore, HA + FA increased LRWC and decreased MP (Table 2). Similarly, it was reported in previous studies that HA + FA applications mitigated the negative impact of heavy metal stress on plant growth properties in lettuce 26 (Haghighi et al. 2010), radish 22 , curly lettuce 9 , triticale 27 , corn 28 and wheat 29 . Humic substances are natural organic polyelectrolytes found in humus, which stabilize organic matter in the soil. Many authors have reported the ability of humic substances to increase the growth of different plant species that grow under different stress conditions 27 . HA + FA positively affect plant growth and yield, directly or indirectly, by improving some physical and chemical properties of the soil. Moreover, HA + FA changes the solubility and bioavailability of toxic heavy metals by forming compounds with them through strong bonds which in turn reduce the heavy metal stress on plant growth 9 . These strong bonds formed with heavy metals are a result of cation exchange capacities (CEC) of humic and fulvic acids. Owing to the high number of carboxyl and phenolic hydroxyl groups in their structure, humic substances have high cation exchange capacities (CEC), which are 600-890 cmol( +)/kg and 1000-1230 cmol( +)/kg for humic acid and fulvic acid, respectively 30,31 . These capacities are 5 to 100 times higher than that of common clay minerals, which makes humic substances perfect stabilizing agents. In addition, although there are various methods and techniques that have been used for stabilization of Cd, Pb, Cu, and Zn, application of humic substances is the only non-chemical and natural method.
In this work, in the absence of Cd stress, no change in the CAT activity was observed due to the application of HA + FA. On the other hand, the effect of Cd stress on CAT activity was found to vary with the concentration of Cd applied for control samples (HA + FA = 0), i.e. decreased when the Cd pollution level was 100 mg/ kg, however increased once the Cd concentration was changed to 200 mg/kg. Although CAT activity reached a very high level in 200 mg/kg Cd polluted control sample (around 90 EU/gr leaf), application of HA + FA at a concentration of 7000 mg/L decreased CAT activity significantly, i.e. to the levels (around 50 EU/gr leaf) even lower than when no Cd stress was present (around 75 EU/gr leaf) ( Table 3).
Our results also showed that, Cd stress conditions increased POD and SOD activity in garden cress. POD activity was increased even more with HA + FA applications whereas and SOD activity was decreased with applications of HA + FA (Table 3).
It has been shown in different studies that various environmental stresses (salinity, water deficit stress and heavy metal stress) affect the enzyme activity 32 . Shao et al. 33 showed that antioxidant activity enhanced under stress conditions. On the other hand, Sergiev et al. 27 suggested that heavy metal applications did not have a significant effect on CAT activity, whereas it increased SOD, GST (glutathione-S-transferase), and GPOX (guaiacol peroxidase) activity in triticale. Similar to our results, Ozkay et al. 9 indicated that heavy metal stress increased SOD activity in curly lettuce, and HA applications supported this increase. The researchers reported that HA applications led to a further increase in SOD and GST activity, which served as a scavenger of reactive oxygen species. This positive effect of HA may most likely be due to the possibility of forming chelating complexes with HA in the nutrient medium or in the plant. Studies showed that humic acid and fulvic acid efficiently immobilize heavy metals and to a larger extent in the mineral soil 34 . Retention capacity was directly related to the amount of added fulvic acid content in the humic substance. The mobility of fulvic acid (FA) is higher due to its smaller size (molecular weight) and higher oxygen content which is twice of HA. In addition, FA is much more reactive because of the presence of many carboxyl (COOH) and hydroxyl (COH) groups (ranges from 520 to 1120 cmol (H +)/kg) in its structure, which makes the exchange capacity of FA more than double that of HA.
The behavior of metals in the soil was affected by humic and fulvic acid application. The effects of HA and FA on Pb, Cd, Ni in soil and availability to plants have been extensively investigated. But, inconsistent findings have been reported due to the complex nature of HA and FA and substantial differences in soil characteristics [35][36][37] .
Earlier reports have indicated that humic and fulvic acid treatments affected plants by affecting the exchangeable nutrient forms in the soil 35 and decreased Pb, and Cd accumulation 38 . They suggested, humic and fulvic acid can be employed to immobilize Pb and Cd in soil. Studies conducted generally focused on the effects of humic acid, while leaving the effects of fulvic acid in humic-made content unevaluated. Previous studies reported the inhibitory effects of humic substances on heavy metal in acidic soils 39 www.nature.com/scientificreports/ humic substances were found to stimulate the metal availability 36,37 . This contradiction can be explained by the unnoticed effect of fulvic acid in the humic content. The underlying reason why the prohibitive impacts of humic substances on metal availability were found at low pH is that the bond formation between metals and fulvic acid dominated under acidic conditions. In fact, in cases where the fulvic acid content in humic compounds is 5% or more, the capacity of heavy metal binding and fixing is particularly high in neutral and light acid soils. On the contrary, humic bits of humic acid content, which is very high compared to fulvic acid content, increases the availability of heavy metal. Thus, it can be said that humic acids are effective for heavy metal bioremediation because humic acids can interrelate with metals to form metal-humic complexes 34 . On the other hand, fulvic acids have been reported to inhibit metal availability and might be employed to decrease metal accumulation in the polluted acidic soils. Humic acid had a stimulating impact on heavy metal presence and were very good for metal bioremediation in alkaline soils 36,37 . As a result, Cd pollution negatively affected plant growth parameters such as plant fresh and dry weight. However, HA + FA applications have been found to reduce this negative effect Considering the plant growth parameters, the best results were determined when HA + FA concentration was 7000 mg/L. We have shown that, it is critical to apply a humic substance with high percentage of FA, which was 10% in this study, to mitigate the adverse effects of heavy metal stress on plant growth.