Environ Sci Pollut Res (2014) 21:13615–13624 DOI 10.1007/s11356-014-3271-3
RESEARCH ARTICLE
Effect of Zn toxicity on root morphology, ultrastructure, and the ability to accumulate Zn in Moso bamboo (Phyllostachys pubescens) Dan Liu & Junren Chen & Qaisar Mahmood & Song Li & Jiasen Wu & Zhengqian Ye & Danli Peng & Wenbo Yan & Kouping Lu
Received: 14 February 2014 / Accepted: 30 June 2014 / Published online: 17 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The effects of zinc (Zn) on seed germination and growth of Moso bamboo (Phyllostachys pubescens) were investigated. Under zinc stress, the seed germination rate did not show significant difference from that of the control. Hydroponics experiments indicated that Moso bamboo had a strong ability to accumulate Zn in the shoot and it reached its maximum value in the shoot at 100 μM Zn. The root Zn concentration ranged from 2,329.29 to 8,642.51 mg kg−1, with the root Zn concentration at 10 μM Zn being 58.23 times that of the control. The root morphology parameters slightly increased at the lower Zn treatments, while growth restriction was evident at higher Zn treatments. Root ultrastructural studies revealed that the cell structure, root tips, and organelles were significantly changed under Zn stress as compared to those of the control. Some abnormalities were evident in the cell walls, vacuoles, mitochondria, plasmalemma, tonoplast, and xylem parenchyma of root cells. While Moso bamboo seems a suitable candidate for phytoremediation, its metal remediation ability should be further explored in future investigations.
Keywords Zn toxicity . Root morphology . Ultrastructure . Phytoremediation . Moso bamboo (Phyllostachys pubescens) Responsible editor: Elena Maestri D. Liu (*) : J. Chen : S. Li : J. Wu : Z. Ye : D. Peng : W. Yan : K. Lu Zhejiang Province Key Laboratory of Carbon Cycling in Forest Ecosystems and Carbon Sequestration, Zhejiang A&F University, Lin’an 311300, Zhejiang, China e-mail: [email protected] Q. Mahmood Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Introduction Consequent to growth of the world economy, soil contamination by heavy metals has emerged as a widespread phenomenon. Though zinc is an essential element for plant metabolic processes, it is considered as one of the heavy metals polluting the environment (Lone et al. 2008). It may be toxic or even lethal to living organisms when present at high concentrations in the environment. About 1,350,000 t of zinc has been released into the environment around the world recently (Singh et al. 2003). Excessive amounts of zinc in soil may cause severe toxicity symptoms in plants like inhibition of seed germination (Michael and Krishnaswamy 2011), delayed or retarded plant growth (Andrade et al. 2009) and slow root development (Lingua et al. 2008), induction of foliar chlorosis (Wang et al. 2009), significant alteration of mitotic activity (Rout and Das 2003), effects on membrane integrity and permeability (Stoyanova and Doncheva 2002) and interference of the solute uptake, transport, osmotic relations, and regulation of essential ions (Cherif et al. 2010). Zinc not only affects plant growth but also gets accumulated in various plant organs, causing various health hazards. Some remedial measures should be adopted to combat zinc toxicity in the environment. Phytoremediation is a new biotechnology which employs hyperaccumulators to remove pollutants from the environment or render them harmless. It has emerged as an alternative technique for removing toxic metals from soil and offers the benefits of being a robust, cost-effective, and environmentally sustainable phenomenon (Islam et al. 2007). The shortcomings of this technique include low yield of hyperaccumulators and slower growth of plants employed in phytoremediation. Furthermore, it is a time-limited process, affected by climatic or seasonal conditions. More than 700 species of metal hyperaccumulators are identified so far; however, only about
13616
18 Zn hyperaccumulator species have been identified at the moment (Reeves and Baker 2000). Furthermore, the best hyperaccumulators should accumulate high levels of contaminants and should exhibit a fast growing ability with high biomass production (Watanabe 1997). Though Moso bamboo (Phyllostachys pubescens) is a nonhyperaccumulator species, it has so many advantages over hyperaccumulators. For example, Moso bamboo grows rapidly, reaching its maximum size within 2 months, with an average height of 15 m (Xu et al. 2011) and the aboveground biomass has been estimated to be 116.5 t DM (dry matter)ha−1 (Shimokawa et al. 2009). It is widely distributed throughout tropical and subtropical zones between the latitudes of 46° N and 47° S (Song et al. 2013). It can survive under the mean annual temperature range of 15–20 °C with a precipitation range of 1,000 to 2,000 mm (Song et al. 2013). The area of its growth in China is more than 3.37 Mha, which is 70 % of the total Chinese bamboo cultivation area (Chen et al. 2009). Moreover, Moso bamboo was found growing well in an old Pb/Zn-mined area, which demonstrated that it has high endurance against heavy metal stress, based on its immense potential as a phytoremediation material. Currently, no reports exist on the heavy metal tolerance and accumulation of Moso bamboo. The aim of the present study was to investigate the effects of zinc on the seed germination and root morphology of Moso bamboo as well as the ultrastructural localization of zinc in Moso bamboo. Another objective was to study the ability of Moso bamboo to accumulate Zn. The study might serve as a theoretical background for the role of Moso bamboo in phytoremediation and its perspective for the reclamation of heavy metal-contaminated soils.
Environ Sci Pollut Res (2014) 21:13615–13624
Hydroponics experiment The experiment was conducted under greenhouse conditions at Zhejiang A&F University in Zhejiang (China) at the geographic coordinates of 30° 19′ N, 119° 35′ E. Seeds were allowed to germinate according to the procedure described for the seed germination experiment. The seeds were sown in a mixture of perlite and vermiculite at 3:1 (v/v), moistened with distilled water, and half strength Yoshida nutrient solution was supplied until seedlings with two leaf pairs were established. After 2 weeks, Moso bamboo seedlings with uniform size were selected based on their similarity in size and transferred to plastic pots containing half strength Yoshida nutrient solution; three plants were grown in each pot. The composition of the nutrient solution was (mg l−1): 57.125 NH4NO3, 25.188 NaH 2PO 4 · 2H 2 O, 44.625 K 2SO 4, 55.375 CaCl 2, 202.5 MgSO4 ·7H2O, 4.663 Na2EDTA, 3.475 FeSO4 ·7H2O, 0.813 MnSO4 ·H2O, 0.0463 (NH4)6MO7O24 ·4H2O, 0.584 H3BO3, 0.0219 ZnSO4 ·7H2O, 0.0194 CuSO4 ·5H2O, 7.438 citric acid (monohydrate), and 0.0625 ml H2SO4. Subsequently, Zn treatments were as follows: (1) 0 μM (control), (2) 10 μM, (3) 25 μM, (4) 50 μM, (5) 100 μM, (6) 200 μM, and (7) 400 μM, with the source of Zn being ZnSO4 ·7H2O. The concentration of zinc in solution existed as Zn cations (Zn2+). The experiment was set according to a random block design, keeping each treatment in triplicate. Plants were grown under glasshouse conditions with natural light, day/ night temperature of 25/30 °C and day/night humidity of 70/ 90 %. The pH of the nutrient solution was adjusted to 5.8 with 0.1 M NaOH or 0.1 M HCl, and it was continuously aerated and renewed every 5th day during the experiment. Plant harvest and elemental analysis
Materials and methods Seed germination experiment Seeds of Moso bamboo were collected from Guilin, Guangxi Province, China. During the seed germination experiment, healthy seeds of the Moso bamboo were surface sterilized in a solution containing 2 g kg−1 KMnO4 for 30 min. After surface sterilization, seeds were thoroughly washed with distilled water. The seeds were sown on a double layer of filter paper in glass Petri dishes (90-mm diameter). The metal treatments in triplicates included 0, 10, 25, 50, 100, 200, and 400 μM with the source of Zn as ZnSO4 ·7H2O. The concentration of zinc in solution was existed as Zn cation (Zn2+). Each Petri plate contained 25 seeds, and the dishes were placed in an incubator at 25 °C. After 10 days, the germination percentage and radical and hypocotyl lengths were recorded.
The Zn-treated plants were harvested on the 30th day. At the time of harvest, the intact plants were washed with distilled water and then immersed in 20 mM Na2EDTA for 15–20 min to remove Zn adhered to the root surfaces. After that, plants were washed thrice with distilled water and finally with deionized water. The parts of plant shoots and roots were separated and their fresh weights were recorded. These plant parts were oven-dried at 70 °C for approximately 72 h and dry weights were recorded. The heavy metal concentrations in Moso bamboo were measured according to the reported methods (Li et al. 2013). The oven-dried plant parts were ground in a stainless steel mill and then passed through a 0.1-mm nylon sieve for Zn analysis. Approximately 0.1 g of the plant sample was digested in the HNO3/HClO4 solution. The digested solutions were washed in 50-ml flasks and volume was made using de-ionized water. The plant Zn concentrations were determined through ICP-MS (Agilent 7500a). The supernatant was
Environ Sci Pollut Res (2014) 21:13615–13624
filtered through a 0.45-μm filter paper, acidified with 15 % HNO3, and finally analyzed for the Zn concentrations by ICP-MS (Agilent 7500a).
13617
Results Effects of Zn on seed germination and hypocotyl and radical lengths
Measurement of root morphology In each replicate, three plants were randomly selected and the root length, root surface area, root volume, and number of root tips of each plant were determined using a root automatism scan apparatus (Epson Expression 10000XL), equipped with the WinRHIZO software. Average values of these three plants in each pot were considered as one replicate. Transmission electron microscopy and scanning electron microscopy Control and treated (200 μM Zn) Moso bamboo plants were selected for the transmission electron microscopy (TEM) studies. The samples were measured according to the existing methods (Islam et al. 2007): small sections of root (1–3 mm in length) were fixed in 4 % glutaraldehyde (v/v) in 0.2 M sodium phosphate buffer (pH 7.2) for 6–8 h and post-fixed by immersion in 1 % OsO4 (osmium(VIII) oxide) for 1 h and finally in 0.2 M phosphate-buffered saline (pH 7.2) for 1–2 h. Dehydration was done in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) followed by acetone, then samples were infiltrated and embedded in Spurr’s resin. Ultra-thin sections (80 nm) were prepared and mounted on copper grids for observation under the transmission electron microscope (JEOL TEM-1200EX) at an accelerating voltage of 60.0 kV. Control and treated (200 μM Zn) Moso bamboo plants were selected for the SEM studies (Hu et al. 2009). Small sections of root (1–3 mm in length) were fixed in 4 % glutaraldehyde (v/v) in 0.2 M sodium phosphate buffer (pH 7.2) for 6–8 h and post-fixed by immersion in 1 % OsO 4 (osmium(VIII) oxide) for 1 h and finally in 0.2 M phosphate-buffered saline (pH 7.2) for 1–2 h. Dehydration was carried out in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) followed by acetone, and then, samples were infiltrated and embedded in Spurr’s resin. In the end, the specimen was dehydrated in a Hitachi Model HCP-2 critical point dryer with liquid CO2. The specimen was coated with gold-palladium in an Eiko Model IB5 ion coater for 4–5 min and then observed in a Hitachi Model TM-1000 SEM. Statistical analyses SPSS statistical package (version 21.0) carried out the statistical analysis. All values reported are means of at least three independent replications. Data were tested at significant levels of P
RESEARCH ARTICLE
Effect of Zn toxicity on root morphology, ultrastructure, and the ability to accumulate Zn in Moso bamboo (Phyllostachys pubescens) Dan Liu & Junren Chen & Qaisar Mahmood & Song Li & Jiasen Wu & Zhengqian Ye & Danli Peng & Wenbo Yan & Kouping Lu
Received: 14 February 2014 / Accepted: 30 June 2014 / Published online: 17 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The effects of zinc (Zn) on seed germination and growth of Moso bamboo (Phyllostachys pubescens) were investigated. Under zinc stress, the seed germination rate did not show significant difference from that of the control. Hydroponics experiments indicated that Moso bamboo had a strong ability to accumulate Zn in the shoot and it reached its maximum value in the shoot at 100 μM Zn. The root Zn concentration ranged from 2,329.29 to 8,642.51 mg kg−1, with the root Zn concentration at 10 μM Zn being 58.23 times that of the control. The root morphology parameters slightly increased at the lower Zn treatments, while growth restriction was evident at higher Zn treatments. Root ultrastructural studies revealed that the cell structure, root tips, and organelles were significantly changed under Zn stress as compared to those of the control. Some abnormalities were evident in the cell walls, vacuoles, mitochondria, plasmalemma, tonoplast, and xylem parenchyma of root cells. While Moso bamboo seems a suitable candidate for phytoremediation, its metal remediation ability should be further explored in future investigations.
Keywords Zn toxicity . Root morphology . Ultrastructure . Phytoremediation . Moso bamboo (Phyllostachys pubescens) Responsible editor: Elena Maestri D. Liu (*) : J. Chen : S. Li : J. Wu : Z. Ye : D. Peng : W. Yan : K. Lu Zhejiang Province Key Laboratory of Carbon Cycling in Forest Ecosystems and Carbon Sequestration, Zhejiang A&F University, Lin’an 311300, Zhejiang, China e-mail: [email protected] Q. Mahmood Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Introduction Consequent to growth of the world economy, soil contamination by heavy metals has emerged as a widespread phenomenon. Though zinc is an essential element for plant metabolic processes, it is considered as one of the heavy metals polluting the environment (Lone et al. 2008). It may be toxic or even lethal to living organisms when present at high concentrations in the environment. About 1,350,000 t of zinc has been released into the environment around the world recently (Singh et al. 2003). Excessive amounts of zinc in soil may cause severe toxicity symptoms in plants like inhibition of seed germination (Michael and Krishnaswamy 2011), delayed or retarded plant growth (Andrade et al. 2009) and slow root development (Lingua et al. 2008), induction of foliar chlorosis (Wang et al. 2009), significant alteration of mitotic activity (Rout and Das 2003), effects on membrane integrity and permeability (Stoyanova and Doncheva 2002) and interference of the solute uptake, transport, osmotic relations, and regulation of essential ions (Cherif et al. 2010). Zinc not only affects plant growth but also gets accumulated in various plant organs, causing various health hazards. Some remedial measures should be adopted to combat zinc toxicity in the environment. Phytoremediation is a new biotechnology which employs hyperaccumulators to remove pollutants from the environment or render them harmless. It has emerged as an alternative technique for removing toxic metals from soil and offers the benefits of being a robust, cost-effective, and environmentally sustainable phenomenon (Islam et al. 2007). The shortcomings of this technique include low yield of hyperaccumulators and slower growth of plants employed in phytoremediation. Furthermore, it is a time-limited process, affected by climatic or seasonal conditions. More than 700 species of metal hyperaccumulators are identified so far; however, only about
13616
18 Zn hyperaccumulator species have been identified at the moment (Reeves and Baker 2000). Furthermore, the best hyperaccumulators should accumulate high levels of contaminants and should exhibit a fast growing ability with high biomass production (Watanabe 1997). Though Moso bamboo (Phyllostachys pubescens) is a nonhyperaccumulator species, it has so many advantages over hyperaccumulators. For example, Moso bamboo grows rapidly, reaching its maximum size within 2 months, with an average height of 15 m (Xu et al. 2011) and the aboveground biomass has been estimated to be 116.5 t DM (dry matter)ha−1 (Shimokawa et al. 2009). It is widely distributed throughout tropical and subtropical zones between the latitudes of 46° N and 47° S (Song et al. 2013). It can survive under the mean annual temperature range of 15–20 °C with a precipitation range of 1,000 to 2,000 mm (Song et al. 2013). The area of its growth in China is more than 3.37 Mha, which is 70 % of the total Chinese bamboo cultivation area (Chen et al. 2009). Moreover, Moso bamboo was found growing well in an old Pb/Zn-mined area, which demonstrated that it has high endurance against heavy metal stress, based on its immense potential as a phytoremediation material. Currently, no reports exist on the heavy metal tolerance and accumulation of Moso bamboo. The aim of the present study was to investigate the effects of zinc on the seed germination and root morphology of Moso bamboo as well as the ultrastructural localization of zinc in Moso bamboo. Another objective was to study the ability of Moso bamboo to accumulate Zn. The study might serve as a theoretical background for the role of Moso bamboo in phytoremediation and its perspective for the reclamation of heavy metal-contaminated soils.
Environ Sci Pollut Res (2014) 21:13615–13624
Hydroponics experiment The experiment was conducted under greenhouse conditions at Zhejiang A&F University in Zhejiang (China) at the geographic coordinates of 30° 19′ N, 119° 35′ E. Seeds were allowed to germinate according to the procedure described for the seed germination experiment. The seeds were sown in a mixture of perlite and vermiculite at 3:1 (v/v), moistened with distilled water, and half strength Yoshida nutrient solution was supplied until seedlings with two leaf pairs were established. After 2 weeks, Moso bamboo seedlings with uniform size were selected based on their similarity in size and transferred to plastic pots containing half strength Yoshida nutrient solution; three plants were grown in each pot. The composition of the nutrient solution was (mg l−1): 57.125 NH4NO3, 25.188 NaH 2PO 4 · 2H 2 O, 44.625 K 2SO 4, 55.375 CaCl 2, 202.5 MgSO4 ·7H2O, 4.663 Na2EDTA, 3.475 FeSO4 ·7H2O, 0.813 MnSO4 ·H2O, 0.0463 (NH4)6MO7O24 ·4H2O, 0.584 H3BO3, 0.0219 ZnSO4 ·7H2O, 0.0194 CuSO4 ·5H2O, 7.438 citric acid (monohydrate), and 0.0625 ml H2SO4. Subsequently, Zn treatments were as follows: (1) 0 μM (control), (2) 10 μM, (3) 25 μM, (4) 50 μM, (5) 100 μM, (6) 200 μM, and (7) 400 μM, with the source of Zn being ZnSO4 ·7H2O. The concentration of zinc in solution existed as Zn cations (Zn2+). The experiment was set according to a random block design, keeping each treatment in triplicate. Plants were grown under glasshouse conditions with natural light, day/ night temperature of 25/30 °C and day/night humidity of 70/ 90 %. The pH of the nutrient solution was adjusted to 5.8 with 0.1 M NaOH or 0.1 M HCl, and it was continuously aerated and renewed every 5th day during the experiment. Plant harvest and elemental analysis
Materials and methods Seed germination experiment Seeds of Moso bamboo were collected from Guilin, Guangxi Province, China. During the seed germination experiment, healthy seeds of the Moso bamboo were surface sterilized in a solution containing 2 g kg−1 KMnO4 for 30 min. After surface sterilization, seeds were thoroughly washed with distilled water. The seeds were sown on a double layer of filter paper in glass Petri dishes (90-mm diameter). The metal treatments in triplicates included 0, 10, 25, 50, 100, 200, and 400 μM with the source of Zn as ZnSO4 ·7H2O. The concentration of zinc in solution was existed as Zn cation (Zn2+). Each Petri plate contained 25 seeds, and the dishes were placed in an incubator at 25 °C. After 10 days, the germination percentage and radical and hypocotyl lengths were recorded.
The Zn-treated plants were harvested on the 30th day. At the time of harvest, the intact plants were washed with distilled water and then immersed in 20 mM Na2EDTA for 15–20 min to remove Zn adhered to the root surfaces. After that, plants were washed thrice with distilled water and finally with deionized water. The parts of plant shoots and roots were separated and their fresh weights were recorded. These plant parts were oven-dried at 70 °C for approximately 72 h and dry weights were recorded. The heavy metal concentrations in Moso bamboo were measured according to the reported methods (Li et al. 2013). The oven-dried plant parts were ground in a stainless steel mill and then passed through a 0.1-mm nylon sieve for Zn analysis. Approximately 0.1 g of the plant sample was digested in the HNO3/HClO4 solution. The digested solutions were washed in 50-ml flasks and volume was made using de-ionized water. The plant Zn concentrations were determined through ICP-MS (Agilent 7500a). The supernatant was
Environ Sci Pollut Res (2014) 21:13615–13624
filtered through a 0.45-μm filter paper, acidified with 15 % HNO3, and finally analyzed for the Zn concentrations by ICP-MS (Agilent 7500a).
13617
Results Effects of Zn on seed germination and hypocotyl and radical lengths
Measurement of root morphology In each replicate, three plants were randomly selected and the root length, root surface area, root volume, and number of root tips of each plant were determined using a root automatism scan apparatus (Epson Expression 10000XL), equipped with the WinRHIZO software. Average values of these three plants in each pot were considered as one replicate. Transmission electron microscopy and scanning electron microscopy Control and treated (200 μM Zn) Moso bamboo plants were selected for the transmission electron microscopy (TEM) studies. The samples were measured according to the existing methods (Islam et al. 2007): small sections of root (1–3 mm in length) were fixed in 4 % glutaraldehyde (v/v) in 0.2 M sodium phosphate buffer (pH 7.2) for 6–8 h and post-fixed by immersion in 1 % OsO4 (osmium(VIII) oxide) for 1 h and finally in 0.2 M phosphate-buffered saline (pH 7.2) for 1–2 h. Dehydration was done in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) followed by acetone, then samples were infiltrated and embedded in Spurr’s resin. Ultra-thin sections (80 nm) were prepared and mounted on copper grids for observation under the transmission electron microscope (JEOL TEM-1200EX) at an accelerating voltage of 60.0 kV. Control and treated (200 μM Zn) Moso bamboo plants were selected for the SEM studies (Hu et al. 2009). Small sections of root (1–3 mm in length) were fixed in 4 % glutaraldehyde (v/v) in 0.2 M sodium phosphate buffer (pH 7.2) for 6–8 h and post-fixed by immersion in 1 % OsO 4 (osmium(VIII) oxide) for 1 h and finally in 0.2 M phosphate-buffered saline (pH 7.2) for 1–2 h. Dehydration was carried out in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) followed by acetone, and then, samples were infiltrated and embedded in Spurr’s resin. In the end, the specimen was dehydrated in a Hitachi Model HCP-2 critical point dryer with liquid CO2. The specimen was coated with gold-palladium in an Eiko Model IB5 ion coater for 4–5 min and then observed in a Hitachi Model TM-1000 SEM. Statistical analyses SPSS statistical package (version 21.0) carried out the statistical analysis. All values reported are means of at least three independent replications. Data were tested at significant levels of P
