Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 37-48, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1827 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Seyedeh Mahsa Hosseini, Sepideh Kalatejari, Mohsen Kafi, Babak Motesharezadeh (2022). Assess- ment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions. Caryologia 75(4): 37-48. doi: 10.36253/caryologia-1827 Received: April 18, 2022 Accepted: December 04, 2022 Published: April 28, 2023 Copyright: © 2022 Seyedeh Mahsa Hos- seini, Sepideh Kalatejari, Mohsen Kafi, Babak Motesharezadeh. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. The data that suppor t the find- ings of this study are openly avail- able in [repository name e.g “Zeno- do”] at http://doi.org/[10.5281/zeno- do.6344758]. Competing Interests: The Author(s) declare(s) no conflict of interest. Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions Seyedeh Mahsa Hosseini1, Sepideh Kalatejari1, Mohsen Kafi2,*, Babak Motesharezadeh3 1 Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Horticultural Science, University College of Agriculture & Natural Resource, University of Tehran, Karaj, Iran 3 Department of Soil Science, Faculty of Agricultural Engineering & Technology, Univer- sity College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran Corresponding author. E-mail: mkafi@ut.ac.ir Abstract. Heavy metals pollution is an important challenge that was cussed by human activity, this stress decreases under salinity. The aim of this study was to investigate the ability of Japanese raisin in the absorption of nitrate (0, 30, and 60 mgL-1) and lead (0, 300, and 600 mgL-1) under salinity stress (0 as control and 3 and 6 dSm-1). Results showed that the studied plant continued to uptake nitrate and potassium under stress conditions of Pb and salinity. Although Na and Cl uptake were observed as a defense mechanism in the plant, the K/Na ratio, and K content increased from 1 to 6 and from 1.8 to 5%, respectively. Also, the most appropriate physiological responses were observed at treatments under contamination level of 300 mg Pb and salinity level of 3 dSm-1, so that the synthesis of malondialdehyde (MDA) and enzymatic activity increased at these levels of HMs and salinity. Based on the results, the studied species were able to uptake moderate concentrations of Pb (34.1-71mg kg-1) under experimen- tal conditions. Hence, its potential for the clean-up of some contaminants in the envi- ronment can be considered by researchers for further research. Novelty statement. This study investigated the cleaning up of some heavy metals and nitrate from the environment and plants’ physiological responses under stressful condi- tions. The plant species (Japanese raisin) characteristics and the results have enough novelty and will be published for the first time. Most hardwood trees such as walnut, oak, beech, poplar, etc. have a slow growth rate. But Japanese raisin tree a new and unknown plant in Iran has very important features. This tree is one of the few trees that in addition to having hardwood, has a very high growth rate. Which can be useful in creating artificial forests, Landscapes, as well as in industrial applications, buildings, and furniture industries. So far, no special research has been done on the phytoreme- diation characteristics of this plant and the selection of this plant in the present study and the study of the ability of this plant to absorb nitrate and lead under salinity stress is a completely new and innovative topic. Keywords: Antioxidant enzymes, Heavy metal, Phytoremediation, Proline, Malondial- dehyde, Salinity. 38 Seyedeh Mahsa Hosseini et al. 1. INTRODUCTION Metal pollution is harmful for human health and the environment. Human activities have been consid- ered an important factor in the contamination of the soil with heavy metals (HMs) (Akinci and Guven 2018; Motesharezadeh et al, 2016). The presence of HMs reduces soil fertility, crop yield, and soil microbial activity (Pinto et al, 2004; Sumiahadi and Acar, 2018). Lead (Pb), a HM pollutant in industrial ecosystems, is important in plant life because of its easily absorption by the plant roots, which is induced by its high accu- mulation in the surface area of the soil (Mosaferi et al, 2008; Wang et al, 2019). In addition to natural pro- cesses, Pb is also produced through the artificial sourc- es (exhaust fumes from automobiles, factories, battery tanks, and pesticides). After Pb is absorbed by roots, it causes changes in metabolic activities of plants, disrupt- ing their growth and development (Sharma and Dubey, 2005; Oguntade et al, 2018). Presence of Pb, leads to a disruption of membrane carriers’ activity of the root cells, depleting nutrients such as magnesium, calcium, and iron. As a result of an experiment, deficiency symp- toms of these nutrients were reported in Pb-treated plants (Sharma and Dubey, 2005). Moreover, the over- use of nitrate fertilizers in agriculture fields leads to nitrate pollution of ground and surface waters (Castro‐ Rodríguez et al, 2016). Climate change and water deficit is the important challenge in agriculture activities all over the world and soil salinity is the one of most important problem that cause by these challenges (Isayenkov and Maathuis, 2019). There are many studies have reported that salin- ity stress induced by NaCl restricts agriculture and crop yield (Isayenkov and Maathuis, 2019). Plant resist- ance to salinity depends on some mechanisms such as antioxidants activity, ion homeostasis, biosynthesis of osmolytes, and gene expression. Phytoremediation is a useful technic based on the living plant’s ability to absorb ionic compound by their roots or leaves and clean up soil, air and water contamination (Berti and Cunningham, 2000). There are many reports on phy- toremediation, such as phytoremediation of high lev- els of nitrate with poplar trees (Castro‐Rodriguez et al, 2016), zinc (Zn) and Pb nitrates with sunflowers (Adeso- dun et al, 2010), nitrate with Salvinia molesta (Ng and Chan, 2017) and Zn, Cd, and Pb with Typha angustifo- lia and Eichhornia crassipes (Sricoth et al, 2018). Gener- ally, stresses such as salinity and heavy metals in which salinity increases the uptake of heav y metals, occur simultaneously in the environment. The results of a study indicated that the presence of Cd along with NaCl in the root environment of four barley cultivars signifi- cantly reduced soil Cd concentration and increased its uptake by plants (Huang et al, 2007). Similarly, Abbasi et al. (2013) investigated the effect of irrigation water salin- ity on the rate of heavy metals uptake in Potamogeton berchtoldi and reported that the concentration of heavy metals (Pb and Cd) in plant increased with the increas- ing salinity up to 4 and 6 dSm-1, respectively. In general, based on the results of the numerous studies, it can be concluded that under conditions of HMs (such as Pb and Cd) and salinity stresses, the plant’s nutritional needs for nutrients such as N, P and K will increase (Khosh- goftarmanesh, 2010; Yan et al, 2020). In fact, the recom- mendation for more application of these nutrients under stress conditions, is a strategic management to prevent reducing plant dry matter. It should be mentioned that nitrate, in principle, increases the resistance to salinity, which has been similarly reported in several studies (Bai et al, 2021). However, there are limited reports on the phytore- mediation ability of Japanese raisin or its ability to grow in the contaminated soils. Based on this background, this study was aimed to assess the potential use of Japa- nese raisin for the phytoremediation of a nitrate/Pb pol- luted soil under conditions of salt stress. 2. MATERIAL AND METHODS 2.1. Plant material and growth condition One-year old seedlings of Japanese raisin (Hove- nia dulcis L.) were prepared from Hirkania greenhouse (Nowshahr, Mazandaran, Iran). Seedlings were grown in plastic bags (25×30 cm), and fertilized with NPK fertiliz- er and a Hoagland based solution during the test period (Motesharezadeh et al, 2016), also the average tempera- ture and the humidity were 25 °C and 70%, respectively (Ramesh et al, 2006). The seedlings were kept for five months and then harvested. Due to the stress induced by moving seedlings from a distant place in the north of the country, it was necessary to apply some treat- ments to plants reinforcement (Table S11). Therefore, to improve the seedlings growth and before the applica- tion of experimental treatments, complete fertilizer and Hoagland nutrient solution were used and also leaching was considered. 1 Supplementary data. 39Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions 2.2. Experimental design To execute the experiment, a neutral culture media (a combination of 70% non-enriched cocopeat and 30% perlite) was used. In order to investigate the capability of Japanese raisin for the phytoremediation of a NO3- / Pb polluted soil under conditions of NaCl stress, a facto- rial greenhouse trial was arranged in a completely ran- domized design with four replications. The treatments consisted of (1) NaCl salinity as the primary factor at three levels of 0 (control), 3, and 6 dSm-1, according to previous reports (Salimi et al, 2012); (2) Nitrate derived from potassium nitrate, as the secondary factor, was applied at three levels (0, 30, and 60 mgL-1) (Gheshlaghi et al, 2015); and (3) Pb form the source of lead nitrate (0, 300, and 600 mgL-1) (Shabani et al, 2015) as the third factor. To have only the effect of nitrate, equivalent of potassium added in the treatments (from the source of potassium nitrate), potassium sulfate was added to oth- er pots. In order to avoid the negative effect of possible stress on plant, potassium nitrate was applied during the holding period every two weeks via irrigation water. 2.3. Biochemical measurements 2.3.a Lead content measurement Dry ashing method was used to analyze plant sam- ples and measure Pb (Wallis, 1996). During the method, to measure metals, plants organic matter is destroyed with controlled heat. Based on the method, one gram of dried and powdered sample of the plant was poured into a crucible and placed in an electric oven at 550 degrees for 4 hours. After leaving the crucibles out of the furnace and reaching ambient temperature, samples were trans- ferred to small beakers using 10 ml of 2 M hydrochloric acid. Then, the beakers were placed on an electric stove until the first white vapors appeared. Next, after reach- ing the ambient temperature, the contents of beakers were filtered through a filter paper inside a 100 ml volu- metric flask and made up to volume with distilled water. Finally, Pb concentrations were reported in samples extract by use of ICP-OES (Inductively Coupled Plasma Atomic Spectroscopy). Pb uptake was measured and reported by multiplying its concentration by the plant dry matter (Sharma et al, 2012). 2.3.b Total Nitrogen and Nitrate content measurement Total concentration of nitrogen (N) in plant sam- ples was measured by Kjeldahl method (Horneck and Miller,1998). In this method ground plant material were digested to H2SO4 at high temperatures by the help of metal catalyst. Total nitrogen of plant was changed to ammonium (NH4+), which is then by titration the con- centration of N was quantified. To measure the nitrate of the plant samples, ion-selective electrode method was used (Miller, 1988). 2.3.c Potassium/sodium ratio and chlore content meas- urements Sodium and potassium concentrations were deter- mined based on the method of wet digestion with hydrochloric acid using flame photometer ELEA (Ryan et al, 2002). In order to measure the chlore (Cl) content in the plant material (Liu, 1998), after extraction, the Cl in the filtrate was analyzed using the colorimetric method on the TRAACS 800TM Auto-Analyzer. In this method, the sample is mixed with the color reagent and dialyzed into the color reagent again. The procedure is based on the release of thiocyanate ions from mercuric thiocy- anate by Cl ions in the sample. The liberated thiocyanate reacts with ferric iron to form a red color complex of fer- ric thiocyanate. The color of the resulting solution is sta- ble and directly proportional to the original Cl concen- tration. The color complex is measured at 480 nm using a 10-mm flow cell. Nitrite (NO2), sulfide, cyanide, thio- cyanate, bromide, and iodine ions cause interferences when present in sufficient amounts. 2.4. Antioxidant enzymes activity measurement In order to evaluate the effect of salinity, nitrate and lead contamination stresses on plant physiologi- cal responses, the changes in enzymatic activity were measured. Among plant enzymes, catalase (CAT) and superoxide dismutase (SOD) are considered as sensitive enzymes indicating plant resistance mechanisms under stress conditions (Khadem Moghadam et al, 2016). Hence, these two enzymes were selected for this goal of the present study. Total protein content was determined following the method described by Bradford (1976). Pro- tein  content was determined using spectrophotometry  at a wavelength of  595 nm. Also, the modified method of Chance and Maehly (1955) was used to measure the activity of peroxidase (POD) enzyme. The activity of superoxide dismutase, the basis of which is its ability to inhibit the photochemical reduction of nitro blue tetra- zolium (NBT), was determined according to the method described by Dhindsa et al. (1981). 40 Seyedeh Mahsa Hosseini et al. 2.5. Soluble carbohydrate content measurement The method of Irigoyen et al. (1992), was used to measure soluble carbohydrates. For this purpose, leave sample were ground in liquid nitrogen, 100 mg of them were blended with 5 mL of 70 % ethanol (wv-1) for 5 min, then centrifuged at 3500 rpm for 10 min at 4 °C. After that 200 mL of the supernatant were added to 1 mL of an anthrone solution, then the absorbance was read by UV/vis spectrophotometer at 625 nm. 2.6. Malondialdehyde level assessment Malondialdehyde (MDA) concentration was meas- ured by thiobarbituric acid method by spectrophotometry. Its concentration was calculated using the extinction coef- ficient MDA-TBA for the complex (Dandekar et al., 2002). 2.7. Proline content measurement To measure proline level, the procedure of Bate et al. (1973) were used. First, 10 ml of acid sulfuric were added to 100 mg of fresh leaf, they were then passed through filter paper. After 2ml ninhydrin and 2ml acid acetic gla- cial were added to 2 ml of extract and kept in benmary counter for 1 h, then toluene was added to them. After 2 h the supernatant was extracted. The absorbance at 520 nm was recorded. 2.8. Lipid peroxidization measurement To quantify the amount of this enzyme, the modi- fied Chance and Maehly (1955) method was used. In this method, 1 ml of potassium phosphate buffer (pH = 6.7) was poured into the cuvette and 17.6 μl of hydro- gen peroxide and 17.6 μl of leaf extract were added to it. The resulting solution was immediately read in a spec- trophotometer at a wavelength of 240 nm for 2 minutes at intervals of 15 seconds to calculate the activity of this enzyme according to the amount of light absorption. 2.9. Statical analysis The present study was executed based on a factorial trial arranged in a completely randomized design (CRD) with four replications. Data were analyzed using SAS 9.2 and MSTATC software. Differences between treatments were determined following Duncan’s Multiple Range Test (DMRT), (P ≤ 0.05). figures were drawn using Excel 2010 software. 3. RESULTS 3.1. Biochemical content measurements 3.1.a. Plants lead level According to the variance analysis results, the inter- actions of salinity, NO3-, and Pb significantly affected Pb concentration (Table S2). Based on the means compari- son results, salinity at levels of 3 and 6 dSm-1 reduced Pb content by 13% and 36%, respectively; nitrate at lev- els of 30 and 60 mgL-1 decreased Pb content by 11% and 12%, respectively; and Pb at levels of 300 and 600 mgL-1 increased Pb content by 45% and 53%, respectively. Also, the highest Pb content (69 mg kg.-1) belonged to the treatments of S1N0Pb2 and S0N0Pb2, and the lowest one was observed at treatments with no added Pb (Fig. 1). To better understand the ability of the studied plant how cope with stress and phytoremediation, the uptake rate was calculated for different treatments (Fig. 2). Results showed that in high salt concentration the high uptake of lead was observed in high nitrate concentra- tion. Accordingly, Pb uptake and accumulation can be considered as a reliable indicator for phytoremediation under salinity stress conditions. 3.1.b. Plants total nitrogen and nitrate level Regarding nitrogen content, it is understandable that there was a significant (P≤0.01) interaction between salin- ity, NO3-, and Pb. Additionally, NO3- significantly affected shoot nitrogen content (P≤0.01) (Table S2). The applica- tion of 30 and 60 mgL-1 NO3- increased N content by 10% and 18%, respectively. The highest N content (7.3%) was m c a m e b m ef c m d a m g ef m g f m i jk m k h m ij i 0 10 20 30 40 50 60 70 80 P b0 P b2 P b1 P b0 P b2 P b1 P b0 P b2 P b1 P b0 P b2 P b1 P b0 P b2 N0 N1 N2 N0 N1 N2 N0 N1 N2 control S1 S2 P b (m g. kg -1 ) Figure 1. Lead (Pb) content in Japanese raisin in response to salin- ity (S0: Control, S1: 3 and S2: 6 dSm-1), nitrate (N0: 0, N1: 30 and N2: 60 mgL-1), and Pb (Pb0: 0, Pb1: 300 and Pb2: 600 mgL-1). Val- ues in each group followed by the same letter are not significantly different according to DMRT at P≤0.05 41Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions observed in S0N2Pb0 and S2N1Pb2 treatments, while the lowest N content (3.3%) was recorded for S1N0Pb2. Based on the results, the interaction of salinity, NO3-, and Pb, significantly affected shoot NO3- content (Table S2). NO3- content reduced by 11% and 21% with the application of 3 and 6 dSm-1 salinity. However, applica- tion of 30 and 60 mgL-1 of NO3- increased NO3- content by 24% and 36%, respectively. The interaction between all three treatments showed that the highest NO3- con- tent (0.61%) was recorded for the treatment of S0N1Pb0, while the lowest NO3- content (0.05%) was observed at the treatment of S2N0Pb2. 3.3.c Plants sodium, potassium and chlore level In accordance with the obtained results, the inter- actions of salinity, NO3-, and Pb significantly (P≤0.01) affected shoot potassium content (Table S2). The lowest values of shoot K concentration were observed at salin- ity treatments of 3 and 6 dSm-1, which were reported to be 1.85% and 1.95%, respectively; while NO3- application at levels of 30 and 60 mgL-1 increased K by 4% and 22%, respectively. The interaction between the treatments indicated that the highest shoot K concentration (5.05%) belonged to the treatment of S0N2Pb0, that was almost 3 times more than the lowest one at treatment of S1N1Pb0, S1N0Pb0 and S2N0Pb2 (1.85%). As results showed, the interactions of salinity, NO3-, and Pb significantly (P≤0.01) affected shoot Na con- centration (Table S2). Considering to the data, it can be found that shoot Na concentration increased up to the 39% by salinity application of 3 or 6 dSm-1, while NO3- application at levels of 30 and 60 mgL-1 decreased shoot Na content by 26% and 25%, respectively. Based on the interactions of studied factors, the highest shoot Na concentration )2.77% (belonged to the treatments of S2N0Pb0, S2N1Pb0 that was fivefold of the S2N2Pb2 treatment as the lowest one. Considering to the K/Na ratio shown in Table 2, it can be found that the accumulation of K effectively con- trolled salinity stress. The highest ratio was recorded in the SIN0Pb1treatment that was six times more than S2N0Pb2 treatment as lowest one. According to variance analysis results it is clear that the interactive effects of salinity, NO3-, and Pb, sig- nificantly affected Cl concentration (Table S2). Also, the results of means comparison of indicated that salinity at levels of 3 and 6 dSm-1 increased Cl content by 17% and 30%, respectively; Nitrate at levels of 30 and 60 mgL- 1 increased Cl content by 9% and 2%, respectively; and Pb at levels of 300 and 600 mgL-1 increased Cl content by 4.9% and 6.8%, respectively. The highest (4.7%) and the lowest (1.4%) values of Cl content were recorded for the treatments of S2N1Pb2 and S0N0Pb0, respectively. High Cl concentration was observed in the treatments with high salinity and nitrate concentration. 3.2. Antioxidant enzymes activity Results showed salinity significantly affected the activities of antioxidant enzymes (Table S3), so that the levels of 3 and 6 dSm-1 increased the activity of POD by 11% and 6%, respectively, while they reduced the activi- ties of SOD by 2 and 15% and CAT by 17 and 63% (Table 1), respectively. Additionally, NO3- at levels of 30 and 60 mgL-1 significantly decreased the activities of POD by 12% and 20%, SOD by 9% and 24%, and CAT by 20% and 15% (Table 1), respectively. Furthermore, Pb at levels of 300 and 600 mgL-1 significantly increased the activities of POD by 12% and 11% and CAT by 27% and 34%, respec- tively, while they reduced the activity of SOD by 39% and 50%, respectively (Table 1). Considering to the results, it can be found that the highest enzymatic activity of SOD, POD and CAT, which are the best indicators of assessing stress conditions, were observed at treatments of 3 dSm- 1 salinity + 30 mgL-1 nitrate (without Pb), 3 dSm-1 salin- ity + 600 mgL-1 Pb (without nitrate) and 600 mgL-1 Pb + 30 mgL-1 nitrate application (without salinity), respec- tively. (Table 1). In other words, with increasing the stud- ied stresses levels including Pb contamination up to 600 mg, salinity up to 6 dSm-1 and nitrate up to 60 mgL-1, the enzymatic activity reduced indicating the reduction of plant defense mechanisms under severe stress conditions. Generally, the relationships between biochemical traits and nutrients status can provide a clear under- h h h h c d h d a h h h h e e h f e h h h h b g h d f 0 0,5 1 1,5 2 2,5 3 Pb0 Pb2 Pb1 Pb0 Pb2 Pb1 Pb0 Pb2 Pb1 Pb0 Pb2 Pb1 Pb0 Pb2 N0 N1 N2 N0 N1 N2 N0 N1 N2 control S1 S2 )1- Sh oo t l ea d up ta ke (m g po t Figure 2. Shoot lead (Pb) uptake in Japanese raisin in response to salinity (S0: Control, S1: 3 and S2: 6 dSm-1), nitrate (N0: 0, N1: 30 and N2: 60 mgL-1), and Pb (Pb0: 0, Pb1: 300 and Pb2: 600 mgL-1). Values in each group followed by the same letter are not significant- ly different according to DMRT at P≤0.05 42 Seyedeh Mahsa Hosseini et al. standing of positive and negative correlations among whole studied parameters. For this purpose, the correla- tion between traits were calculated. The results showed the high positive correlation between proline and MDA by R2=0.879, N and K by R2= 0.718, soluble carbohydrate and MDA by R2= 0.612 and lipid peroxidase and MDA by R2= 0.574 (Table 2). 3.3. Soluble carbohydrates content To evaluate the biochemical and physiological responses of plant against studied stresses, soluble car- bohydrates were measured. Based on the results, the most values of soluble carbohydrates were reported at treatments of S1N0Pb2 and S0N0Pb2, indicating that HMs stress had more effect on this trait in comparison with salinity. The highest content of soluble carbohy- drate was recorded in S0N0Pb2 that was 3 time more than S2N2Pb2 as lowest treatment. 3.4. Malondialdehyde level of plants Also, there was a significant difference in proline concentration among different studied treatments com- pared to control. Malondialdehyde was measured as an important indicator of plant response to abiotic stresses. Results showed, the most values of this parameter were observed at moderate levels of Pb application (with- out salinity) and also the synthesis of this biochemical product significantly reduced by the expansion of HMs, salinity and nitrate stress. Lowest value of MDA was recorded in S2N2Pb2 treatment that show this param- eter were deceased in high salt, nitrate and lead concen- tration. 3.5. Proline level of plants The most values of proline were observed at mod- erate levels of Pb application (without salinity) and also the synthesis of this biochemical product significantly reduced by the expansion of HMs, salinity and nitrate stress. The highest value of proline was recorded in the S0N0 treatment that was twofold higher than S2N2 treatment as last one. 3.6. Lipid peroxidation The increasing trend was observed for lipid peroxi- dation and the most values of this parameter belonged to the treatments with low levels of stress (without salin- ity and without nitrate). Highest content was observed in the S0N0 treatment, while the treatment with high level of nitrogen and lead ranked last treatment. 4. DISCUSSION 4.1. Biochemical traits affected by different level of nitrate and lead under salinity stress 4.1.a lead uptake and concentration were affected under different nitrate level Based on the results, Pb pollution caused more salinity (Na) uptake. In other words, salinity can increase HMs stress, which means the intensifica- tion of the stress induced by salinity and also has been Table 1. Activities of antioxidant enzyme in response to treatments. Salinity (dSm-1) Nitrate (mgL-1) Pb (mgL-1) POD SOD CAT Control 0 0 0.52d* 161.9c 5kl 300 0.29lmn 82.15ijk 56.37cd 600 0.36hij 87.87ij 21.83gh 30 0 0.19p 68.63lmn 3.01l 300 0.35 h-k 93.02i 54.08cd 600 0.38ghi 128.4ef 86.26a 60 0 0.45ef 120.7fg 30.8f 300 0.56cd 140.1d 58.77c 600 0.58c 117.5g 67.41b 3 0 0 0.34ijk 67.05mn 72.25b 300 0.34ijk 132.9de 50.19d 600 0.8a 175.5b 55.34cd 30 0 0.54cd 188.5a 43.41e 300 0.69b 154.3c 11.05jk 600 0.31klm 59.99no 9.76jkl 60 0 0.23op 52.44o 53cd 300 0.27mno 74.59klm 10.55jkl 600 0.33jkl 86.29ij 14.98hij 6 0 0 0.48e 115.2gh 3.52jl 300 0.57c 132.3de 25.93fg 600 0.36hij 105.6h 27.79fg 30 0 0.42fg 106.3h 14.84hij 300 0.36hij 79.4jkl 12.66ij 600 0.38ghi 89.69ij 20.79gh 60 0 0.23op 49.88o 8jkl 300 0.39gh 114.4gh 18.7hi 600 0.26no 58.77no 10.07jkl * Means within a column followed by the same letters are not signifi- cantly different at P ≤ 0.05 according to Duncan’s multiple range test. 43Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions considered by many researchers. Generally, when the stress inhibits plant growth and reduces transpiration, the increase in contaminant uptake will be stopped or reduced. Accordingly, in the present experiment, the salinity stress without HMs contamination, led to reduce contaminant (Pb) uptake. The critical level of Pb con- tamination in soil, considered by researchers, is 50 mg kg-1 soil (Prasad, 2004). Also, the normal range of Pb in plant tissues is between 0.2 to 20 mg kg-1, but its critical and contamination levels in plant is more than 20 mg kg-1, reducing the yield and plants dry matter (Alloway, 1990). Accordingly, high concentrations (34.6-71.6 mg kg -1) of Pb accumulated in treatments of Pb contami- nation indicating the ability of the studied species for HMs phytoremediation. In fact, the studied plant has an appropriate potential for phytoremediation under condi- tions of simultaneous stresses. 4.1.b N and Nitrate level improved the plant resistance under salinity stress The percentage of N and NO3 and the accumulation of K, Na and Cl represent the intensity of plant response to the studied stresses (HMs and salinity). Results indi- cated that salinity or Na content reduced by supplying nitrate. It should be mentioned that nitrate increases plants resistance to salinity, which also has been report- ed in numerous researches (Kafkafi et al, 1992). Some researchers believe that reduction of nitrate concen- tration is because of the negative interaction between Cl and nitrate and antagonistic effect of Cl on nitrate uptake. While, others attribute it to the plant’s response under saline conditions reducing water uptake (Lauter and Munns, 1986). The results of a study showed that phytoremediation of HVs (Co, Cu, Cr, Ni, and Pb) pol- lution by aquatic hyacinth was only effective at high con- centrations of nitrate and by decreasing nitrate concen- tration the phytoremediation efficiency decreased (Tan- gahu et al, 2011; Bai et al, 2021). Loska and Wiechula (2003) reported that the presence of any type of contam- inants in water and soil resources led to pollutants accu- mulation in plant organs, changing enzymatic activities. Similarly, the results of the present study are consistent with those of recent studies (Dayani et al, 2009; Huse- jnovic et al, 2018). 4.1.c High potassium to sodium ratio improve phytoex- traction in contract to Chlore Khoshgoftar et al. (2004) reported that HMs uptake from the soil solution increased with the increase of NaCl level, while no such effect was observed for NaNO3. It is assumed that chloride ion positively affect- ed HMs (Pb and Cd) solubility in soil and their uptake by plants. Generally, chloride ion increases the dynam- ics and adsorption capacity of the metals. Having high salinity tolerance, is another important strategy of plants for resistance against salinity. For example, grasses and Atriplex/Salicornia are capable to grow at the salinity levels up to 1.2-1.7 dSm-1 and 21-28 dSm-1, respectively. Table 2. Pearson correlation coefficients between characteristics. N NO3 K Na K/Na Cl Pb Con. Pb Uptake Sol. Carb Proline MDA Lipid POX SOD CAT N 1 NO3- 0.495** 1 K 0.768** 0.575** 1 Na -0.093ns 0.071ns 0.049ns 1 K/Na 0.468** 0.359** 0.520** -0.721** 1 Cl 0.497** 0.409** 0.412** 0.104ns 0.249* 1 Pb Con. 0.340** 0.158ns 0.344** -0.331** 0.259* 0.145ns 1 Pb Uptake -0.209ns -0.217ns -0.198ns -0.174ns 0.086ns 0.060ns -0.149ns 1 Sol. Carb -0.084ns 0.083ns -0.065ns 0.215ns -0.140ns 0.022ns -0.484** 0.008ns 1 Proline -0.006ns 0.179ns 0.189ns 0.366** -0.180ns -0.094ns -0.249* -0.222* 0.504** 1 MDA -0.126ns 0.064ns -0.054ns 0.009ns 0.016ns -0.136ns -0.418** 0.042ns 0.818** 0.612** 1 Lipid 0.056ns 0.215ns 0.195ns 0.302ns -0.067ns -0.071ns -0.234* -0.288** 0.475** 0.879** 0.574** 1 POX -0.221* -0.146ns -0.032ns 0.259* -0.293** 0.131ns -0.205ns 0.165ns 0.256* 0.106ns 0.122ns 0.004ns 1 SOD -0.113ns -0.140ns -0.085ns 0.037ns 0.067ns -0.285** 0.218ns 0.073ns 0.328** 0.158ns 0.363** 0.206ns 0.386** 1 CAT -0.213ns -0.270* -0.099ns -0.299** 0.163ns -0.240* -0.050ns -0.219* 0.202ns 0.302** 0.373** 0.276* -0.081ns 0.222* 1 ** represent significant difference at P ≤ 0.01, * represent significant difference at P ≤ 0.05, n.s represent no significant difference 44 Seyedeh Mahsa Hosseini et al. Regarding to the results of the present study, it seems that the Japanese raisin (Hovenia dulcis) can be considered as a relatively tolerant species at moderate salinity levels due to its appropriate responses to salin- ity levels of 3 and 6 dSm-1, the accumulation of K and Na and also the high ratio of K/Na in different stress treatments. In addition to salinity stress, Pb contami- nation also has a specified critical level based on soil and plant studies. Potassium accumulation is probably a defense mechanism and increasing the ratio of K/Na is a strategic way for resistance to salinity stress (Kib- ria and Hoque, 2015). Generally, these results indicat- ed the intensity of the effect of stress treatments (HMs and salinity) on the one hand and the plant resistance responses to Pb and salinity. Also, Kibria and Hoque (2015) conducted a field experiment to investigate the effect of the mitigation of soil salinity on rice by appli- cation of K and Zn fertilizers. The results demonstrated that K+/Na+ ratio in the grains significantly affected by the application of K. Therefore, it may be induced by the fact that the application of higher doses of K and Zn fertilizers could alleviate the adverse effects of salinity in rice via increasing nutrient uptake and maintaining a higher K+/Na+ ratio. Boudaghi Malidareh et al. (2014) reported a significant relationship between the amount of K fertilizer and HMs pollution (cadmium concen- tration) in the soil. Furthermore, the results of a study demonstrated that soil salinity can be improved by the application of nitrogen and potassium fertilizers (Shan- ker, 2005). Azari et al. (2005) investigated the role of potassium on nitrate and Cd contamination in potato and onion. They found that the concentrations of nitrate and cadmium in potato and onion tubers significantly decreased following the application of potassium and zinc fertilizers, and the highest nitrate and Cd contami- nation was recorded for the treatment with the unbal- anced fertilizer use. Under salinity stress, the concentra- tions of potassium and phosphorus in the stem signifi- cantly decreased, while the concentration of sodium in the leaves increased. The similar results were also report- ed by Khalilpoor and Jafarinia (2017) and Yousefinia and Ghasemiyan (2016). 4.2. Antioxidant enzymes activity was affected by different levels of HM and salt stress It seems that the increase in enzymatic activity is one of the main strategies tolerating HMs contamina- tion and salinity stresses (Malar et al, 2016). Accord- ingly, the changes in activity of SOD enzyme are con- sidered as the appropriate indicators of stress manage- ment. The results of the present study showed that the increased SOD activity under stress conditions was due to the plant’s survival on the one hand and contaminants purification on the other hand. However, the simulta- neous increase in the stresses of HMs contamination, salinity and nitrate, up to the maximum levels, reduced the activity of all three studied enzymes. Malar et al. (2016) reported that plants use different mechanisms to cope with HMs toxicity. Alizadeh (2012) reported that the contamination lead and cadmium disrupted the growth of two poplar species and plant biomass signifi- cantly reduced at high levels of pollutants. Furthermore, the reduced vegetative growth caused by HMs stress in plants may be because of the suppressed activities of catalase and superoxide dismutase (Schutzendubel and Polle, 2002). Jabeen and Ahmad (2012) reported that salinity increased the activity of peroxidase but decreased that of catalase. Similar results were also reported on canola (Abili and Zare, 2014) and maize (AbdElgawad et al, 2016). Michalak (2006) has found that antioxidant enzymes can scavenge reactive oxygen species (ROS) when the plant grows under HMs stress. Also, Verma and Dubey (2003) observed that Pb toxicity changed the activity of antioxidant enzymes in rice plants. Barandeh and Kavousi (2017) reported that the activities of antioxi- dant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase significantly increased in lentil seedlings with the increasing in Cd concentration. Simi- larly, Hendry et al. (1992) illustrated that HMs are also a reason for oxidative stress via the production of free radi- cals of reactive oxygen which can react with lipids and finally lead to the lipid peroxidation, membrane dam- age, and enzymatic inactivation (Dixi et al, 2001). Simi- lar results were reported by Verma and Dubey (2003) and AbdElqawad et al. (2016). Generally, it has been reported that stress increases the activities of antioxidant enzymes (Meloni et al, 2003) but at an intolerable intensity obvi- ously reduces their activities (Amiriyan Mojarad et al, 2018). The results of the present study in agreement with previous reports showed the increment of SOD, POD and CAT by increasing the HM and salt stresses level. 4.3. Soluble carbohydrate was affected by different levels of HM and salt stress Soluble carbohydrate content was affected by differ- ent nitrate level and stress condition. The results of cur- rent study showed the decreasing trend by increasing salt and lead level. Weisany et al. (2014) reported that at all three growth stages (pre-flowering, post-flowering, and seed filling), salinity stress decreased shoot fresh and dry weights, plant yield, root and leaf also soluble carbohydrate content of these tissue in soybean, but the 45Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions application of zinc fertilizer alleviated these negative effects. Decreased biomass production induced by HMs stress may be because of a disturbance in uptake and transmission of nutrients and water into the aerial parts of plants (Sudova and Vosatka, 2007). 4.4. Malondialdehyde synthesis were changed under HM and salt stress The synthesis of biochemical compounds such as malondialdehyde has been considered as another impor- tant mechanism to withstand stress conditions. The stress-adapted plants seem to be more capable to syn- thesis these metabolites. There are numerous studies confirming the increased synthesis of malondialdehyde and some plant biochemical/enzymatic compounds as a response to stress conditions (Juknys et al, 2012; Aljahali and Alhassan, 2020). Based on the results of the pre- sent study, there was a significant and negative correla- tion between shoot Pb concentration with MDA synthe- sis and also between the activity of POX with the K/Na ratio. The similar results have been reported by Aljahali and Alhassan (2020). 4.5. Proline level affected under stress condition Proline is a non-enzymatic antioxidant known as bio-marker that showed plants response to the stress (Petrovic et al, 2020). In the water caltrop plant, they suggested proline accumulation as a good biomarker of HMs stress (Petrovic et al, 2020; Bi, et al., 2021; Duan, et al., 2022; Guo, et al, 2021; Guo, et al, 2022). The results of proline accumulation, showed the decreasing trend by increasing the nitrate concentration under high level of lead. These results indicated the positive effect of nitrate to deceasing the side effect of HMs and salt stress as mentioned before. Similarly, Bai et al, (2021) suggested that phytoremediation of HVs pollution by sweet sor- ghum was only effective at high concentrations of nitrate and by decreasing nitrate concentration the phytoreme- diation efficiency decreased. 4.6. Lipid peroxidation were affected under stress condition Heavy metals pollution, causes changes in some plant processes such as lipid peroxidation (Ashraf et al, 2017). The results of this study showed the high lipid per- oxidation under control treatment. Similar results were reported on rice, that by increasing lead level the lipid peroxidation was decreased (Ashraf et al, 2017; Li, et al, 2021; Sun, et al. 2021; Xu, et al, 2021; Zhang, et al. 2022 ). 5. CONCLUSIONS Based on the obtained results of the present study, it can be concluded that with the increasing in salinity stress from 0 to 3 dSm-1, the content of N (from 5.69% to 5.26%), K (from 4% to 3.21%) and nitrate (from 0.36 to 0.28 mg kg-1) significantly reduced. Also, results showed that with the increasing in salinity stress (from 3 to 6 dSm-1) and nitrate level (from 30 to 60 mgL-1) in the soil, plant Pb concentration significantly decreased. Additionally, under conditions of Pb stress, the uptake of nutrients (especially macronutrients) significantly improved with the increasing in the nitrate level from 30 to 60 mgL-1. It appears that stress conditions increased plant’s nitrate requirement, which could be considered as a strategy for the improvement of plant tolerance under HMs stress. On the other hand, the increase in K ranged from 1.8 to 5% and also K/Na ratio ranged from 1 to 6 can be considered as a resistance mechanism of plants under salinity stress. 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