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Abstract ［Objectives］ This study was conducted to discuss the Pb-enrichment ability of Iris pseudacorus, and reveal the effects of different concentrations of Pb stress on the physiological metabolism of I. pseudacorus.
［Methods］I. pseudacorus L. as a test material was cultured by the nutrient solution culture method and analyzed for malondialdehyde (MDA), glutathione (GSH), cysteine (Cys) and proline (Pro) contents of plant leaves as well as catalase (CAT) and superoxide dismutase (SOD) activity under different concentrations of Pb stress (0, 200, 400, 600, 800, 1 000, 1 200 mg/L).
［Results］ With the increase of Pb concentration, the Pb content of leaves and roots gradually increased, and the order of increase was root>leaf. Roots and leaves reached maximums at the high concentration of 1 200 mg/L Pb, and their contents were 9 034.6 and 11.2 mg/kg, respectively. Under Pb stress, the contents of GSH, Cys and Pro, and the activity of CAT and SOD in I. pseudacorus showed a trend of first increasing and then decreasing with the increase of Pb concentration, and each index reached a maximum under the treatment of 400 mg/L. The level of MDA in the leaves of I. pseudacorus rose significantly under Pb stress, indicating that Pb stress caused the cell membrane of I. pseudacorus to be damaged by peroxidation.
［Conclusions］This study provides a theoretical basis for further research and use of this plant for metal pollution remediation in the future.
Key words Iris pseudacorus; Lead stress; Accumulation; Physiology
Received: June 21, 2021 Accepted: September 24, 2021
Supported by Key R&D Project of Shanxi Province (201903D221070); Applied Basic Research Project of Shanxi Academy of Agricultural Sciences (YBSJJ2017).
Song WANG (1983-), male, P. R. China, assistant research fellow, devoted to research about physiological ecology and landscape planning and design of garden plants.
*Corresponding author.
Plants are an open system. In the natural environment, drought, waterlogging, soil salinization, heavy metal pollution, diseases and pests and other stressful environmental factors often adversely affect plant metabolism and growth. In particular, the increasingly serious soil pollution of heavy metals has to a certain extent become an important factor that restricts plant growth and regional distribution, and affected the yield and quality of crops. The continuous accumulation of harmful heavy metals in the soil is one of the negative factors that lead to soil degradation and the reduction of crop yield and quality, which directly leads to harm to human life and health［1-2］. Therefore, the prevention and control of soil toxic heavy metal pollution has become a hotspot and focus of research in the international environmental field. Lead (Pb) is one of the most abundant heavy metal elements that pollute the environment and endanger the growth of organisms［3］. Pb easily combines with organic matter in the soil to form insoluble substances, which generally accumulate in the topsoil layer of the soil. The large accumulation of Pb in plant tissues will cause the disturbance of active oxygen metabolism in the body, lead to lipid peroxidation of cell membranes, and ultimately affect the normal growth of plants［4］. Pb also has serious damage to the human nervous system, bone marrow hematopoietic system, digestive system, immune system, kidney and reproductive system［5］. Therefore, carrying out research on the physiological resistance mechanism of plants to Pb, screening out heavy metal soil pollution remediation plants with strong resistance and good ornamental properties and using the adsorption and accumulation effect of plants on lead to reduce the lead concentration in the soil environment is of great significance to the research on the prevention and control of heavy metal lead pollution.
For this reason, in this study, Iris pseudacorus L. as a test material was cultured by the solution culture method and analyzed for malondialdehyde (MDA), glutathione (GSH), cysteine (Cys) and proline (Pro) contents of plant leaves as well as catalase (CAT) and superoxide dismutase (SOD) activity under Pb stress, and Pb absorption and accumulation and distribution of I. pseudacorus under the stress of different Pb concentrations were studied, aiming to explore the lead-enrichment capacity of I. pseudacorus, reveal the effects of different concentrations of Pb stress on the physiological metabolism of I. pseudacorus, and provide a theoretical basis for further research and use of this plant for metal pollution restoration in the future.
Materials and Methods
Tested materials
The test material was I. pseudacorus. The plant material was cultivated in the Iris germplasm nursery of College of Horticulture, Shanxi Agricultural University (Institute of Horticulture), and the seeds were natural and fruitful seeds of an asexually-reproduced population.
Experimental methods
The experiment was conducted in the College of Horticulture, Shanxi Agricultural University. I. pseudacorus seeds were disinfected with 0.5% NaClO for 20 min, rinsed with tap water for several times, and then soaked to accelerate germination. After the seeds germinated, the seeds with the same germination were selected and sown in petri dishes, and 4 weeks later, the seedling plants with the same growth were selected and placed in 4 L plastic boxes for solution culture. The containers were covered with 1 cm thick perforated white foam boards, and the rhizomes of the seedlings were fixed in the holes of the foam boards with sponges. 1/2 of Hoagland nutrient solution was changed with equal amount of refresh solution every 2 d, and each box was planted with 6 seedlings. Pb was added in the form of PbNO3 after 8 weeks of pre-culture, and the concentrations were: 0 (treatment 1), 200 (treatment 2), 400 (treatment 3), 600 (treatment 4), 800 (treatment 5), 1 000 (treatment 6), and 1 200 (treatment 7), a total of 7 treatments, and each treatment was repeated 3 times. The same amount of nutrient solution was supplemented every 4 d, and samples were taken 20 d after Pb treatment, rinsed with tap water and distilled water, and measured for the Pb concentrations of roots and leaves and the physiological and biochemical indexes of leaves after absorbing the surface water. Index determination methods
The determined indexes included lead content (inductively coupled plasma atomic emission spectrometry)［6］, malondialdehyde (MDA) content (thiobarbituric acid colorimetry)［7］, superoxide dismutase (SOD) activity (NBT light reduction method)［8］, catalase (CAT) (ultraviolet absorption method)［9］, proline content (Pro) (ninhydrin colorimetric method)［10］, and glutathione (GSH) and cysteine (Cys) contents, the last two of which were determined referring to the method of Tian［6］.
Data processing
Excel 2010 software was used to calculate the experimental data and draw graphs, and SPSS19.0 was used for complete multiple comparisons (Duncan), with different letters indicate significant differences between data (P<0.05).
Results and Analysis
Enrichment and distribution of Pb in different organs of I. pseudacorus
It can be seen from the changes of Pb contents accumulated in the leaves and roots of I. pseudacorus under different concentrations of Pb in Fig. 1 that the Pb contents in the leaves and roots of I. pseudacorus basically increased with the increase of Pb concentration, and the absorbed Pb mainly accumulated in the roots, showing an order of root>leaf. The accumulation amount of the root system at the low concentration of 200 mg/L Pb was 556.4 mg/kg, which was quite different from the control; and at the high concentration of 1 200 mg/L Pb, the maximum value was reached at 9 034.6 mg/kg, which was 16.2 times that of the 200 mg/L Pb treatment. The condition of the leaves was similar to roots. At low concentrations, the amount of lead accumulation was relatively small. The Pb content was only 5.74 mg/kg at 200 mg/L Pb, and reached at the high concentration of 1 200 mg/L Pb, a maximum value of 11.2 mg/kg, which was significantly different from the control, showing an increasing trend.
Effects of lead stress on antioxidant enzyme activity of I. pseudacorus
It can be seen from Fig. 2 that the activity of SOD and CAT of I. pseudacorus under Pb stress increased slightly and then decreased. For the increases of SOD and CAT activity under various Pb treatment concentrations, specifically, the values were slightly higher in stress treatments 200 and 400 mg/L than the control, and showed that the higher the Pb stress concentration, the higher the activity of SOD and CAT, while when the concentration was greater than 400 mg/L, the activity of SOD and CAT of I. pseudacorus showed a downward trend. The results showed that the antioxidant enzymes SOD and CAT of I. pseudacorus could induce resistance to low concentration Pb stress, while the stress of high concentration Pb obviously inhibited the activity of SOD and CAT. Effects of Pb stress on the content of sulfur compounds in I. pseudacorus
It can be seen from Fig. 3 that the contents of GSH and Cys in the leaves showed a trend of first increasing and then decreasing with the increase of the Pb stress concentration, and reached maximums under the 400 mg/L Pb stress treatment, which were respectively, 0.233 and 3.353 μmol/g, which were significantly different from the control, 1.10 times and 1.06 times that of the control, respectively. It indicated that the ability of I. pseudacorus to synthesize GSH and Cys increased with the increase of Pb stress concentration within a certain range, and the stress of 400 mg/L Pb concentration might be more conducive to inducing the production of GSH and Cys in plants, while after the Pb stress concentration exceeded 400 mg/L, the GSH and Cys contents of I. pseudacorus showed a downward trend, and the Cys content under the stress of 1 200 mg/L Pb concentration was lower than that of the control, and was 98.9% of the control. It showed that high concentration of Pb stress could inhibit the synthesis of Cys substance in I. pseudacorus, but it still had a certain promoting effect on the production of GSH.
Song WANG et al. Physiological and Biochemical Responses of Iris pseudacorus to Lead Stress
Effects of Pb stress on the contents of Pro and MDA of I. pseudacorus
It can be seen from Fig. 4 that after Pb stress, the content of Pro in the leaves of I. pseudacorus was significantly different from that of the control. The Pro content in the leaves of I. pseudacorus was the largest under the treatment of 400 mg/L Pb, and was 18.09 times that of the control. With the further increase of Pb concentration, the Pro content began to decline, but high Pb stress still promoted Pro synthesis to a certain extent. It indicated that low-concentration Pb stress could obviously induce the increase of Pro content in I. pseudacorus, while high-concentration Pb stress could weaken such effect. The results in Fig. 4 showed that with the increase of Pb concentration, the MDA content in the leaves of I. pseudacorus showed a gradually increasing trend. The MDA contents under various stress treatments were significantly different from that of the control. Among them, the MDA content reached a maximum value under the stress of 1 200 mg/L Pb, which was 5.482 mmol/g, 19.12 times that of the control, indicating that the growth of I. pseudacorus had been significantly inhibited at this concentration, Discussion and Conclusions
For lead-enriched plant Sedum alfredii under the treatment of 160 mg/L Pb, the maximum lead contents of the shoots and roots were 514 and 13 922 mg/kg, respectively［11］. The roots of Phaseolus vulgaris in hydroponic nutrition added with 1 mmol/L Pb accumulated Pb up to 75 000 mg/kg［12］. For Iris lactea under the stress of 10 mmol/L Pb, the Pb content in the shoots and roots reached 3 332 and 8 844 mg/kg, respectively, showing strong enrichment ability, i.e., it is a potential Pb hyper-enrichment plants［13］. Under hydroponics plus Pb stress, the maximum absorption and enrichment of Pb concentration in the roots and leaves of I. pseudacorus reached 9 034.6 and 11.2 mg/kg, respectively, which met the condition that Pb content in leaves or aboveground parts (dry weight) of plants reaches more than 1 000 μg/g. Since the S. alfredii was adult plants, which had a large biomass, especially the thick rhizomes, which accumulated certain nutrients, which might play a very important role in plant growth and development, reproduction, and resistance to adversity. I. pseudacorus in this study was seedlings from seed germination, which had small biomass, and the Pb content of each part of I. pseudacorus was thus much less than that of S. alfredii.
The absorption, accumulation and distribution of Pb by I. pseudacorus were quite different. The absorption was mainly accumulated in the root system, and the order was root system>leaf. The root-to-leaf transfer rate reached a maximum value at 200 mg/L Pb, which was only 1.03%. Panich-pat et al.［14］ obtained the same result when studying Typha angustifolia. They believed that the content of Pb in roots was much higher than that in leaves, and the concentration of Pb in leaves did not depend on the treatment concentration of Pb. There might be limiting factors in the transport of Pb from roots to leaves.
Thiol (-SH) has a strong affinity with heavy metal ions. The metabolism and physiological effects of non-protein thiol substances such as GSH and Cys containing thiol are closely related to the detoxification of heavy metals in plants and have received wide attention［15］. In this study, with the increase from 0 to 400 mg/L under single Pb stress, the GSH and Cys contents of the leaves of I. pseudacorus showed an increasing trend and produced significant differences, which is consistent with the research results of Sun et al.［16］ and Gupta et al.［17］ on S. alfredii, indicating that I. pseudacorus might synthesize a large amount of non-protein thiol compounds under Pb stress to reduce the toxicity of Pb stress to it. Yuan et al.［18］ also proposed that Pb stress induced a large amount of GSH synthesis, and by analyzing the correlation between the content of Pb and the contents of GSH and Cys in roots and leaves of I. lactea under different Pb treatments, they found that there was a significant positive correlation between the content of Pb and the content of GSH, indicating that GSH plays an important role in Pb detoxification. However, this study also found that the Cys content under the stress of 1 200 mg/L Pb was lower than the control, which might be because Cys was consumed as a substrate for the synthesis of GSH, resulting in low Cys content and indirectly reducing Pb damage through GSH. The free radicals produced by the Pb stress-induced biological metabolism process have a harmful effect on plant cell membranes, leading to a significant increase in the content of membrane lipid peroxidation product MDA. MDA can also cross-link with proteins, nucleic acids, amino acids and other active substances to form insoluble compound deposits and further interfere the normal life activities of cells［19］. Organisms’ own protective enzyme system can scavenge free radicals and reduce harm［20］. The results of this study showed that the MDA content of I. pseudacorus under relatively low concentration of 200-600 mg/L Pb was slightly different from the control, indicating that the cell membrane of I. pseudacorus leaves was not significantly damaged at such concentration, and their integrity and functionality were good. The reason might be that under the stress of Pb concentration lower than 400 mg/L, the antioxidant activity of SOD and CAT and the content of Pro in the leaves of I. pseudacorus increased, which improved the resistance to Pb stress of I. pseudacorus. However, with the increase of the Pb stress concentration, especially under the relatively high Pb stress of 800-1 200 mg/L, although I. pseudacorus itself induced a slight increase in Pro content through the stress, the antioxidant activity of SOD and CAT still decreased and the content of MDA increased significantly. It indicated that the damage caused by high concentrations of Pb stress had exceeded the protective ability of I. pseudacorus to resist stress, and Pro, as an osmotic regulator of cell membrane, might be one of the important regulators that increase Pb stress resistance of I. pseudacorus.
References
［1］ ODJEGBA VJ, FASIDI IO. Accumulation of trace elements by Pistia stratiotes: Implications for phytoremediation［J］. Ecotoxicology, 2004(13): 637-646.
［2］ QIN TC, WU YS, WANG HX, et al. Effect of cadmium, lead and their interactions on the physiological and ecological characteristics of root system of Brassica chinensis［J］. Acta Ecologica Sinica, 1998, 18(3): 320-325. (in Chinese)
［3］ HAN YL, YUAN HY, HUANG SZ, et al. Cadmium tolerance and accumulation by two species of Iris［J］. Ecotoxicology, 2007(16): 557-563.
［4］ CHO UH, PARK JO. Mercury-induced oxidative stress in tomato seedlings［J］. Plant Science, 2000(159): 1-9.
［5］ MCLAUGHIIN MJ, PARKER DR, CLARKE JM. Metals and micronutrients-food safety issues［J］. Field Crops Research, 1999(60): 143-163. ［6］ TIAN SQ, ZHU XD, YUAN HY, et al. Effect of Pb stress on Pb accumulation and non-protein thiol content in Louisiana Iris［J］. Northern Horticulture, 2015(21): 77-82. (in Chinese)
［7］ WENG M, CUI L, LIU F, et al. Effects of drought stress on antioxidant enzymes in seedlings of different wheat genotypes［J］. Pakistan Journal of Botany, 2015, 47(1): 49-56.
［8］ ZHANG F, WANG Y, LOU Z, et al. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza)［J］. Chemosphere, 2007, 67(1): 44-50.
［9］ ZHANG SQ, LI Y, WU WH. Plant physiological experiment technology course［M］. Beijing: Beijing Science Press, 2011. (in Chinese)
［10］ BATES LS, WALDREN RP, TEARE ID. Rapid determination of free proline for water-stress studies［J］. Plant and Soil, 1973(39): 205-207.
［11］ HE B, YANG X, NI W, et al. Sedum alfredii: A new lead-accumulating ecotype［J］. Acta Botanica Sinica, 2001, 44(11): 1365-1370.
［12］ PIECHALAK A, TOMASZEWSKA B, BARALKIEWICZ D, et al. Accumulation and detoxification of lead ions in legumes［J］. Phytochemistry, 2002, 60(2): 153-162.
［13］ HAN YL, HUANG SZ, GU JG, et al. Tolerance and accumulation of lead by species of Iris L.［J］. Ecotoxicology, 2008, 17(8): 853-859.
［14］ PANICH-PAT T, POKETHITIYOOK P, KRUATRACHUE M, et al. Removal of lead from contaminated soils by Typha angustifolia［J］. Water Air and Soil Pollution, 2004, 155(1-4): 159-171.
［15］ GAO KH, GE Y, ZHANG CH. Effects of sulfur starvation on the non-protein thiol content and glutathione S-transferase activity of rice seedlings under cadmium stress［J］. Chinese Journal of Applied Ecology, 2011, 22(7): 1796-1802. (in Chinese)
［16］ SUN Q, WANG C. Effects of soil exogenous Cd and Pb compound pollution on the synthesis of phytochelatin and glutathione in the roots of wheat (Triticum aestivum L.)［J］. Ecology and Environment, 2008, 17(5): 1833-1838. (in Chinese)
［17］ GUPTA DK, HUANG HG, YANG XE, et al. The detoxification of lead in Sedum alfredii H. is not related to phytochelatins but the glutathione［J］. Journal of Hazardous Materials, 2010, 177(1-3): 437-444.
［18］ YUAN HY, GUO Z, TONG HY, et al. Effects of exogenous glutathione on Pb accumulation and non-protein thiol content in Iris lactea var. chinensis under Pb stress［J］. Journal of Soil and Water Conservation, 2013, 27(4): 212-216. (in Chinese)
［19］ XU QS, SHI GX, DU KH. Effect of Cd2+ on antioxidase system and ultrastructure of Ottelia alismoides (L.) Pers. Leaves［J］. Rural Eco-environment, 2001, 17(2): 30-34. (in Chinese)
［20］ LI SZ, ZHENG HZ, CHEN JM, et al. Effects of environmental factors on the stem sap flow［J］. Journal of Anhui Agricultural Sciences, 2008, 36(25): 10758-10759. (in Chinese)
［Methods］I. pseudacorus L. as a test material was cultured by the nutrient solution culture method and analyzed for malondialdehyde (MDA), glutathione (GSH), cysteine (Cys) and proline (Pro) contents of plant leaves as well as catalase (CAT) and superoxide dismutase (SOD) activity under different concentrations of Pb stress (0, 200, 400, 600, 800, 1 000, 1 200 mg/L).
［Results］ With the increase of Pb concentration, the Pb content of leaves and roots gradually increased, and the order of increase was root>leaf. Roots and leaves reached maximums at the high concentration of 1 200 mg/L Pb, and their contents were 9 034.6 and 11.2 mg/kg, respectively. Under Pb stress, the contents of GSH, Cys and Pro, and the activity of CAT and SOD in I. pseudacorus showed a trend of first increasing and then decreasing with the increase of Pb concentration, and each index reached a maximum under the treatment of 400 mg/L. The level of MDA in the leaves of I. pseudacorus rose significantly under Pb stress, indicating that Pb stress caused the cell membrane of I. pseudacorus to be damaged by peroxidation.
［Conclusions］This study provides a theoretical basis for further research and use of this plant for metal pollution remediation in the future.
Key words Iris pseudacorus; Lead stress; Accumulation; Physiology
Received: June 21, 2021 Accepted: September 24, 2021
Supported by Key R&D Project of Shanxi Province (201903D221070); Applied Basic Research Project of Shanxi Academy of Agricultural Sciences (YBSJJ2017).
Song WANG (1983-), male, P. R. China, assistant research fellow, devoted to research about physiological ecology and landscape planning and design of garden plants.
*Corresponding author.
Plants are an open system. In the natural environment, drought, waterlogging, soil salinization, heavy metal pollution, diseases and pests and other stressful environmental factors often adversely affect plant metabolism and growth. In particular, the increasingly serious soil pollution of heavy metals has to a certain extent become an important factor that restricts plant growth and regional distribution, and affected the yield and quality of crops. The continuous accumulation of harmful heavy metals in the soil is one of the negative factors that lead to soil degradation and the reduction of crop yield and quality, which directly leads to harm to human life and health［1-2］. Therefore, the prevention and control of soil toxic heavy metal pollution has become a hotspot and focus of research in the international environmental field. Lead (Pb) is one of the most abundant heavy metal elements that pollute the environment and endanger the growth of organisms［3］. Pb easily combines with organic matter in the soil to form insoluble substances, which generally accumulate in the topsoil layer of the soil. The large accumulation of Pb in plant tissues will cause the disturbance of active oxygen metabolism in the body, lead to lipid peroxidation of cell membranes, and ultimately affect the normal growth of plants［4］. Pb also has serious damage to the human nervous system, bone marrow hematopoietic system, digestive system, immune system, kidney and reproductive system［5］. Therefore, carrying out research on the physiological resistance mechanism of plants to Pb, screening out heavy metal soil pollution remediation plants with strong resistance and good ornamental properties and using the adsorption and accumulation effect of plants on lead to reduce the lead concentration in the soil environment is of great significance to the research on the prevention and control of heavy metal lead pollution.
For this reason, in this study, Iris pseudacorus L. as a test material was cultured by the solution culture method and analyzed for malondialdehyde (MDA), glutathione (GSH), cysteine (Cys) and proline (Pro) contents of plant leaves as well as catalase (CAT) and superoxide dismutase (SOD) activity under Pb stress, and Pb absorption and accumulation and distribution of I. pseudacorus under the stress of different Pb concentrations were studied, aiming to explore the lead-enrichment capacity of I. pseudacorus, reveal the effects of different concentrations of Pb stress on the physiological metabolism of I. pseudacorus, and provide a theoretical basis for further research and use of this plant for metal pollution restoration in the future.
Materials and Methods
Tested materials
The test material was I. pseudacorus. The plant material was cultivated in the Iris germplasm nursery of College of Horticulture, Shanxi Agricultural University (Institute of Horticulture), and the seeds were natural and fruitful seeds of an asexually-reproduced population.
Experimental methods
The experiment was conducted in the College of Horticulture, Shanxi Agricultural University. I. pseudacorus seeds were disinfected with 0.5% NaClO for 20 min, rinsed with tap water for several times, and then soaked to accelerate germination. After the seeds germinated, the seeds with the same germination were selected and sown in petri dishes, and 4 weeks later, the seedling plants with the same growth were selected and placed in 4 L plastic boxes for solution culture. The containers were covered with 1 cm thick perforated white foam boards, and the rhizomes of the seedlings were fixed in the holes of the foam boards with sponges. 1/2 of Hoagland nutrient solution was changed with equal amount of refresh solution every 2 d, and each box was planted with 6 seedlings. Pb was added in the form of PbNO3 after 8 weeks of pre-culture, and the concentrations were: 0 (treatment 1), 200 (treatment 2), 400 (treatment 3), 600 (treatment 4), 800 (treatment 5), 1 000 (treatment 6), and 1 200 (treatment 7), a total of 7 treatments, and each treatment was repeated 3 times. The same amount of nutrient solution was supplemented every 4 d, and samples were taken 20 d after Pb treatment, rinsed with tap water and distilled water, and measured for the Pb concentrations of roots and leaves and the physiological and biochemical indexes of leaves after absorbing the surface water. Index determination methods
The determined indexes included lead content (inductively coupled plasma atomic emission spectrometry)［6］, malondialdehyde (MDA) content (thiobarbituric acid colorimetry)［7］, superoxide dismutase (SOD) activity (NBT light reduction method)［8］, catalase (CAT) (ultraviolet absorption method)［9］, proline content (Pro) (ninhydrin colorimetric method)［10］, and glutathione (GSH) and cysteine (Cys) contents, the last two of which were determined referring to the method of Tian［6］.
Data processing
Excel 2010 software was used to calculate the experimental data and draw graphs, and SPSS19.0 was used for complete multiple comparisons (Duncan), with different letters indicate significant differences between data (P<0.05).
Results and Analysis
Enrichment and distribution of Pb in different organs of I. pseudacorus
It can be seen from the changes of Pb contents accumulated in the leaves and roots of I. pseudacorus under different concentrations of Pb in Fig. 1 that the Pb contents in the leaves and roots of I. pseudacorus basically increased with the increase of Pb concentration, and the absorbed Pb mainly accumulated in the roots, showing an order of root>leaf. The accumulation amount of the root system at the low concentration of 200 mg/L Pb was 556.4 mg/kg, which was quite different from the control; and at the high concentration of 1 200 mg/L Pb, the maximum value was reached at 9 034.6 mg/kg, which was 16.2 times that of the 200 mg/L Pb treatment. The condition of the leaves was similar to roots. At low concentrations, the amount of lead accumulation was relatively small. The Pb content was only 5.74 mg/kg at 200 mg/L Pb, and reached at the high concentration of 1 200 mg/L Pb, a maximum value of 11.2 mg/kg, which was significantly different from the control, showing an increasing trend.
Effects of lead stress on antioxidant enzyme activity of I. pseudacorus
It can be seen from Fig. 2 that the activity of SOD and CAT of I. pseudacorus under Pb stress increased slightly and then decreased. For the increases of SOD and CAT activity under various Pb treatment concentrations, specifically, the values were slightly higher in stress treatments 200 and 400 mg/L than the control, and showed that the higher the Pb stress concentration, the higher the activity of SOD and CAT, while when the concentration was greater than 400 mg/L, the activity of SOD and CAT of I. pseudacorus showed a downward trend. The results showed that the antioxidant enzymes SOD and CAT of I. pseudacorus could induce resistance to low concentration Pb stress, while the stress of high concentration Pb obviously inhibited the activity of SOD and CAT. Effects of Pb stress on the content of sulfur compounds in I. pseudacorus
It can be seen from Fig. 3 that the contents of GSH and Cys in the leaves showed a trend of first increasing and then decreasing with the increase of the Pb stress concentration, and reached maximums under the 400 mg/L Pb stress treatment, which were respectively, 0.233 and 3.353 μmol/g, which were significantly different from the control, 1.10 times and 1.06 times that of the control, respectively. It indicated that the ability of I. pseudacorus to synthesize GSH and Cys increased with the increase of Pb stress concentration within a certain range, and the stress of 400 mg/L Pb concentration might be more conducive to inducing the production of GSH and Cys in plants, while after the Pb stress concentration exceeded 400 mg/L, the GSH and Cys contents of I. pseudacorus showed a downward trend, and the Cys content under the stress of 1 200 mg/L Pb concentration was lower than that of the control, and was 98.9% of the control. It showed that high concentration of Pb stress could inhibit the synthesis of Cys substance in I. pseudacorus, but it still had a certain promoting effect on the production of GSH.
Song WANG et al. Physiological and Biochemical Responses of Iris pseudacorus to Lead Stress
Effects of Pb stress on the contents of Pro and MDA of I. pseudacorus
It can be seen from Fig. 4 that after Pb stress, the content of Pro in the leaves of I. pseudacorus was significantly different from that of the control. The Pro content in the leaves of I. pseudacorus was the largest under the treatment of 400 mg/L Pb, and was 18.09 times that of the control. With the further increase of Pb concentration, the Pro content began to decline, but high Pb stress still promoted Pro synthesis to a certain extent. It indicated that low-concentration Pb stress could obviously induce the increase of Pro content in I. pseudacorus, while high-concentration Pb stress could weaken such effect. The results in Fig. 4 showed that with the increase of Pb concentration, the MDA content in the leaves of I. pseudacorus showed a gradually increasing trend. The MDA contents under various stress treatments were significantly different from that of the control. Among them, the MDA content reached a maximum value under the stress of 1 200 mg/L Pb, which was 5.482 mmol/g, 19.12 times that of the control, indicating that the growth of I. pseudacorus had been significantly inhibited at this concentration, Discussion and Conclusions
For lead-enriched plant Sedum alfredii under the treatment of 160 mg/L Pb, the maximum lead contents of the shoots and roots were 514 and 13 922 mg/kg, respectively［11］. The roots of Phaseolus vulgaris in hydroponic nutrition added with 1 mmol/L Pb accumulated Pb up to 75 000 mg/kg［12］. For Iris lactea under the stress of 10 mmol/L Pb, the Pb content in the shoots and roots reached 3 332 and 8 844 mg/kg, respectively, showing strong enrichment ability, i.e., it is a potential Pb hyper-enrichment plants［13］. Under hydroponics plus Pb stress, the maximum absorption and enrichment of Pb concentration in the roots and leaves of I. pseudacorus reached 9 034.6 and 11.2 mg/kg, respectively, which met the condition that Pb content in leaves or aboveground parts (dry weight) of plants reaches more than 1 000 μg/g. Since the S. alfredii was adult plants, which had a large biomass, especially the thick rhizomes, which accumulated certain nutrients, which might play a very important role in plant growth and development, reproduction, and resistance to adversity. I. pseudacorus in this study was seedlings from seed germination, which had small biomass, and the Pb content of each part of I. pseudacorus was thus much less than that of S. alfredii.
The absorption, accumulation and distribution of Pb by I. pseudacorus were quite different. The absorption was mainly accumulated in the root system, and the order was root system>leaf. The root-to-leaf transfer rate reached a maximum value at 200 mg/L Pb, which was only 1.03%. Panich-pat et al.［14］ obtained the same result when studying Typha angustifolia. They believed that the content of Pb in roots was much higher than that in leaves, and the concentration of Pb in leaves did not depend on the treatment concentration of Pb. There might be limiting factors in the transport of Pb from roots to leaves.
Thiol (-SH) has a strong affinity with heavy metal ions. The metabolism and physiological effects of non-protein thiol substances such as GSH and Cys containing thiol are closely related to the detoxification of heavy metals in plants and have received wide attention［15］. In this study, with the increase from 0 to 400 mg/L under single Pb stress, the GSH and Cys contents of the leaves of I. pseudacorus showed an increasing trend and produced significant differences, which is consistent with the research results of Sun et al.［16］ and Gupta et al.［17］ on S. alfredii, indicating that I. pseudacorus might synthesize a large amount of non-protein thiol compounds under Pb stress to reduce the toxicity of Pb stress to it. Yuan et al.［18］ also proposed that Pb stress induced a large amount of GSH synthesis, and by analyzing the correlation between the content of Pb and the contents of GSH and Cys in roots and leaves of I. lactea under different Pb treatments, they found that there was a significant positive correlation between the content of Pb and the content of GSH, indicating that GSH plays an important role in Pb detoxification. However, this study also found that the Cys content under the stress of 1 200 mg/L Pb was lower than the control, which might be because Cys was consumed as a substrate for the synthesis of GSH, resulting in low Cys content and indirectly reducing Pb damage through GSH. The free radicals produced by the Pb stress-induced biological metabolism process have a harmful effect on plant cell membranes, leading to a significant increase in the content of membrane lipid peroxidation product MDA. MDA can also cross-link with proteins, nucleic acids, amino acids and other active substances to form insoluble compound deposits and further interfere the normal life activities of cells［19］. Organisms’ own protective enzyme system can scavenge free radicals and reduce harm［20］. The results of this study showed that the MDA content of I. pseudacorus under relatively low concentration of 200-600 mg/L Pb was slightly different from the control, indicating that the cell membrane of I. pseudacorus leaves was not significantly damaged at such concentration, and their integrity and functionality were good. The reason might be that under the stress of Pb concentration lower than 400 mg/L, the antioxidant activity of SOD and CAT and the content of Pro in the leaves of I. pseudacorus increased, which improved the resistance to Pb stress of I. pseudacorus. However, with the increase of the Pb stress concentration, especially under the relatively high Pb stress of 800-1 200 mg/L, although I. pseudacorus itself induced a slight increase in Pro content through the stress, the antioxidant activity of SOD and CAT still decreased and the content of MDA increased significantly. It indicated that the damage caused by high concentrations of Pb stress had exceeded the protective ability of I. pseudacorus to resist stress, and Pro, as an osmotic regulator of cell membrane, might be one of the important regulators that increase Pb stress resistance of I. pseudacorus.
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