Journal of Integrative Agriculture
July 2012
2012, 11(7): 1129-1136
RESEARCH ARTICLE
Relationship Between Polyamines Metabolism and Cell Death in Roots of Malus hupehensis Rehd. Under Cadmium Stress JIANG Qian-qian1, YANG Hong-qiang1, 2, SUN Xiao-li1, LI Qiang1, RAN Kun1 and ZHANG Xin-rong1 1 2
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, P.R.China State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an 271018, P.R.China
Abstract The free putrescine (Put) content, the hydrogen peroxide (H2O 2) content and the polyamine oxidase (PAO) activity in roots of Malus hupehensis Rehd. var. pinyiensis Jiang (PYTC) were significantly increased, and reached its peak at 1, 2 and 6 h, respectively, under cadmium treatment. The free spermine (Spm) and spermidine (Spd) contents were dramatically decreased, and reached the minimum value at 4-6 h, then remained relatively stable. The change in total free polyamines (PAs) content was consistent with that of free Put. The number of root dead cells was gradually increased after treatment for 24 h, and the typical characteristics of programmed cell death (PCD) were displayed at 48 h. Throughout the Cd treatment process, changes in PAs metabolism appeared to be prior to cell death increase, and the H2O2 content was always maintained at a high level. These results indicated that polyamines could initiate cell death by generating H2O2 in roots of Malus hupehensis Rehd. under CdSO4 stress. Key words: Malus hupehensis Rehd., cadmium, cell death, polyamines metabolism
INTRODUCTION The natural polyamines (PAs) are low molecular weight and aliphatic nitrogenous compounds with high biological activity, widely formed and stored by nearly all eukaryotic cells. The most common free PAs in plants are diamine putrescine (Put), triamine spermidine (Spd) and tetramine spermine (Spm) (Bouchereau et al. 1999). The Spd and Spm can be oxidatively catabolised to peroxide (H2O2) and other metabolites by polyamine oxidase (PAO). In plants, these metabolites and PAs play critical roles in a range of developmental and physiological processes, such as regulation of DNA replication, membrane stability, senescence, cell division, differentiation, and death (Kuthanová et al. 2004; Kuehn
2005; Groppa and Benavides 2008). Cell death is a common phenomenon in the process of life, in which programmed cell death (PCD) is an important ways to resist pathogen infection and adapt to stress (Reape et al. 2008). Cadmium (Cd) is a major heavy metal, which is widely recognized as a serious environmental pollutant. Even at low concentration, it can not only damage several metabolic activities leading to plant growth inhibition and even death, but also constitute a serious threat to the balance of ecological environment and food security. At present, a number of farms and orchards have been suffered Cd contamination at different levels, because of long-term application of phosphorous fertilizer, wastewater irrigation, emissions of industrial and mining waste pollution (Ranieri et al.
Received 13 December, 2012 Accepted 9 September, 2011 JIANG Qian-qian, Tel: +86-538-8249304, E-mail: [email protected]; Correspondence YANG Hong-qiang, Tel: +86-538-8249304, E-mail: [email protected]
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
1130
2007; Lin et al. 2007). Therefore, studies on the damage of Cd to plants are increasing. However, most studies have focused on the effects of Cd on plant growth and development, membrane lipid peroxidation and antioxidant system (Sun et al. 2004; Cheng et al. 2005); little research has been concerned on the changes in PAs metabolism under Cd stress. As a variety of Malus hupehensis Rehd., Malus hupehensis Rehd. var. pinyiensis Jiang (Chinese name is Pingyi Tiancha, PYTC) is a unique resource of apple rootstocks in China, which can be also used as a class of tea-like plant with higher value (Liu et al. 2004; Yang and Shu 2007). Whether used as apple rootstock or for tea, the Cd seriously threatens the growth of PYTC and human health. Zhao and Yang (2008) have found that Cd stress caused changes in endogenous Put, Spm and Spd contents in leaves of PYTC seedlings. But the characteristics of cell death in roots of PYTC under Cd stress are not clear, meanwhile, the relationship between polyamines metabolism and cell death is also worth to explore. Therefore, the aim of this study was to detect cell death and changes in polyamine contents, and provide some evidences to elucidate the mechanism underlined cell death in roots of PYTC seedlings under Cd stress.
JIANG Qian-qian et al.
also reached the highest value at 1 h, and then decreased gradually. The change tendency of total polyamines content was consistent with that of free Put (Fig. 2-A).
RESULTS Effects of Cd on the free polyamine content in roots of PYTC seedlings As shown in Fig. 1-A, the Put content in PYTC roots under Cd stress within 96 h was significantly higher than that of the control. The Put content reached its highest level at 1 h, and then decreased gradually. Within 24-96 h, the change of Put content tended to be smooth, but the content was still significantly higher than that of the control. As shown in Fig. 1-B and C, the Spd and Spm contents in PYTC roots under Cd stress both decreased and reached the minimum at 4-6 h. Thereafter, the Spd content fluctuated slowly and was still lower than the control. Under Cd stress, the total content of free polyamines in PYTC roots increased primly, then decreased, and
Fig. 1 Effects of Cd on the content of free polyamines in roots of PYTC seedlings. A, the content of free Put. B, the content of free Spd. C, the content of free Spm. Each value is mean±SE of three independent experiments and the vertical bars indicate SE (n=3). Values for each treatment level having a common letter(s) are not significantly different at the 5% level by Duncan’s multiple range test. FM, fress mass. The same as below.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Relationship Between Polyamines Metabolism and Cell Death in Roots of Malus hupehensis Rehd. Under
1131
Effects of Cd on the value of (Spd+Spm)/Put in roots of PYTC seedlings
Effects of Cd on the H2O2 content in roots of PYTC seedlings
Under Cd stress, the value of (Spd+Spm)/Put decreased significantly, and was lower than that of the control throughout the treatment. After the initiation of the treatment, the value decreased dramatically at 1 h, and then changed gently and kept at low level, reached the lowest value at 6 h (Fig. 2-B).
As shown in Fig. 3-B, the H 2O 2 content increased dramatically in PYTC roots under Cd stress and reached the highest level at 2 h. Thereafter, the H2O2 content decreased slightly, but was still significantly higher than that of the control and kept at a high level.
Effects of Cd on the cell death in roots of PYTC seedlings As shown in Fig. 4-A, the number of dead cells increased along with the increase of Cd concentration after 48 h treatment. The amount of dead cells was significantly more than that of control when Cd con-
Fig. 2 Effects of Cd on the content of total polyamines and the value of (Spd+Spm)/Put in roots of PYTC seedlings. A, the content of total polyamines. B, the value of (Spd+Spm)/Put.
Effects of Cd on the PAO activity in roots of PYTC seedlings As shown in Fig. 3-A, the PAO activity increased, and reached the highest level at 6 h, then decreased gradually, but it was still significantly higher than that of the control and maintained at a high level.
Fig. 3 Effects of Cd on the activity of PAO and the content of H2O2 in roots of PYTC seedlings. A, the activity of PAO. B, the content of H2O2 .
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
JIANG Qian-qian et al.
1132
tures were kept in normal morphology. The uniform nucleoplasm and clear nucleolus could be observed (Fig. 5-A). After 200 µmol L-1 Cd treatment for 48 h, condensation of chromatin, deformation of nucleus, shrinkage or invagination of nuclear membrane were observed in the nucleus of PYTC extensive roots. The boundaries of nuclear membrane blurred and some parts broken. So the nucleoplasm leaked into the cytoplast. Finally, the nuclei disappeared and the chromatin condensed into a half-moon form, located on the edge (Fig. 5-B and C). As shown in Fig. 5-D, the mitochondrial morphology did not appear abnormal: the mitochondrial structure was clear, the inner and outer membrane and the cristae were all integrated in the extensive roots of PYTC seedlings without Cd stress. After 200 µmol L -1 Cd treatment, mitochondria were distorted and swollen, the structure of outer membrane and cristae became blurred, and the cristae was gradually broken and dissolved, small vacuolation appeared in mitochondria, and the vacuolation expanded until the disintegration of some mitochondria (Fig. 5-E and F). The structural changes in nucleus and mitochondria indicated the occurrence of PCD in roots of PYTC under Cd stress. Fig. 4 Effects of Cd on the cell death in roots of PYTC seedlings. A, treatment of different concentrations Cd for 48 h. B, treatment of 200 µmol L-1 Cd for 0 (control), 0.5, 1, 2, 4, 6, 12, 24, 48, 72, and 96 h.
centration was over 200 µmol L -1. These results showed that, after 48 h treatment, 200 µmol L-1 Cd could increase the membrane permeability then resulted in substantial cell death. However, Fig. 4-B showed that 200 µmol L-1 Cd had no obvious effect on the cell death in the early state of treatment (
July 2012
2012, 11(7): 1129-1136
RESEARCH ARTICLE
Relationship Between Polyamines Metabolism and Cell Death in Roots of Malus hupehensis Rehd. Under Cadmium Stress JIANG Qian-qian1, YANG Hong-qiang1, 2, SUN Xiao-li1, LI Qiang1, RAN Kun1 and ZHANG Xin-rong1 1 2
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, P.R.China State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an 271018, P.R.China
Abstract The free putrescine (Put) content, the hydrogen peroxide (H2O 2) content and the polyamine oxidase (PAO) activity in roots of Malus hupehensis Rehd. var. pinyiensis Jiang (PYTC) were significantly increased, and reached its peak at 1, 2 and 6 h, respectively, under cadmium treatment. The free spermine (Spm) and spermidine (Spd) contents were dramatically decreased, and reached the minimum value at 4-6 h, then remained relatively stable. The change in total free polyamines (PAs) content was consistent with that of free Put. The number of root dead cells was gradually increased after treatment for 24 h, and the typical characteristics of programmed cell death (PCD) were displayed at 48 h. Throughout the Cd treatment process, changes in PAs metabolism appeared to be prior to cell death increase, and the H2O2 content was always maintained at a high level. These results indicated that polyamines could initiate cell death by generating H2O2 in roots of Malus hupehensis Rehd. under CdSO4 stress. Key words: Malus hupehensis Rehd., cadmium, cell death, polyamines metabolism
INTRODUCTION The natural polyamines (PAs) are low molecular weight and aliphatic nitrogenous compounds with high biological activity, widely formed and stored by nearly all eukaryotic cells. The most common free PAs in plants are diamine putrescine (Put), triamine spermidine (Spd) and tetramine spermine (Spm) (Bouchereau et al. 1999). The Spd and Spm can be oxidatively catabolised to peroxide (H2O2) and other metabolites by polyamine oxidase (PAO). In plants, these metabolites and PAs play critical roles in a range of developmental and physiological processes, such as regulation of DNA replication, membrane stability, senescence, cell division, differentiation, and death (Kuthanová et al. 2004; Kuehn
2005; Groppa and Benavides 2008). Cell death is a common phenomenon in the process of life, in which programmed cell death (PCD) is an important ways to resist pathogen infection and adapt to stress (Reape et al. 2008). Cadmium (Cd) is a major heavy metal, which is widely recognized as a serious environmental pollutant. Even at low concentration, it can not only damage several metabolic activities leading to plant growth inhibition and even death, but also constitute a serious threat to the balance of ecological environment and food security. At present, a number of farms and orchards have been suffered Cd contamination at different levels, because of long-term application of phosphorous fertilizer, wastewater irrigation, emissions of industrial and mining waste pollution (Ranieri et al.
Received 13 December, 2012 Accepted 9 September, 2011 JIANG Qian-qian, Tel: +86-538-8249304, E-mail: [email protected]; Correspondence YANG Hong-qiang, Tel: +86-538-8249304, E-mail: [email protected]
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
1130
2007; Lin et al. 2007). Therefore, studies on the damage of Cd to plants are increasing. However, most studies have focused on the effects of Cd on plant growth and development, membrane lipid peroxidation and antioxidant system (Sun et al. 2004; Cheng et al. 2005); little research has been concerned on the changes in PAs metabolism under Cd stress. As a variety of Malus hupehensis Rehd., Malus hupehensis Rehd. var. pinyiensis Jiang (Chinese name is Pingyi Tiancha, PYTC) is a unique resource of apple rootstocks in China, which can be also used as a class of tea-like plant with higher value (Liu et al. 2004; Yang and Shu 2007). Whether used as apple rootstock or for tea, the Cd seriously threatens the growth of PYTC and human health. Zhao and Yang (2008) have found that Cd stress caused changes in endogenous Put, Spm and Spd contents in leaves of PYTC seedlings. But the characteristics of cell death in roots of PYTC under Cd stress are not clear, meanwhile, the relationship between polyamines metabolism and cell death is also worth to explore. Therefore, the aim of this study was to detect cell death and changes in polyamine contents, and provide some evidences to elucidate the mechanism underlined cell death in roots of PYTC seedlings under Cd stress.
JIANG Qian-qian et al.
also reached the highest value at 1 h, and then decreased gradually. The change tendency of total polyamines content was consistent with that of free Put (Fig. 2-A).
RESULTS Effects of Cd on the free polyamine content in roots of PYTC seedlings As shown in Fig. 1-A, the Put content in PYTC roots under Cd stress within 96 h was significantly higher than that of the control. The Put content reached its highest level at 1 h, and then decreased gradually. Within 24-96 h, the change of Put content tended to be smooth, but the content was still significantly higher than that of the control. As shown in Fig. 1-B and C, the Spd and Spm contents in PYTC roots under Cd stress both decreased and reached the minimum at 4-6 h. Thereafter, the Spd content fluctuated slowly and was still lower than the control. Under Cd stress, the total content of free polyamines in PYTC roots increased primly, then decreased, and
Fig. 1 Effects of Cd on the content of free polyamines in roots of PYTC seedlings. A, the content of free Put. B, the content of free Spd. C, the content of free Spm. Each value is mean±SE of three independent experiments and the vertical bars indicate SE (n=3). Values for each treatment level having a common letter(s) are not significantly different at the 5% level by Duncan’s multiple range test. FM, fress mass. The same as below.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Relationship Between Polyamines Metabolism and Cell Death in Roots of Malus hupehensis Rehd. Under
1131
Effects of Cd on the value of (Spd+Spm)/Put in roots of PYTC seedlings
Effects of Cd on the H2O2 content in roots of PYTC seedlings
Under Cd stress, the value of (Spd+Spm)/Put decreased significantly, and was lower than that of the control throughout the treatment. After the initiation of the treatment, the value decreased dramatically at 1 h, and then changed gently and kept at low level, reached the lowest value at 6 h (Fig. 2-B).
As shown in Fig. 3-B, the H 2O 2 content increased dramatically in PYTC roots under Cd stress and reached the highest level at 2 h. Thereafter, the H2O2 content decreased slightly, but was still significantly higher than that of the control and kept at a high level.
Effects of Cd on the cell death in roots of PYTC seedlings As shown in Fig. 4-A, the number of dead cells increased along with the increase of Cd concentration after 48 h treatment. The amount of dead cells was significantly more than that of control when Cd con-
Fig. 2 Effects of Cd on the content of total polyamines and the value of (Spd+Spm)/Put in roots of PYTC seedlings. A, the content of total polyamines. B, the value of (Spd+Spm)/Put.
Effects of Cd on the PAO activity in roots of PYTC seedlings As shown in Fig. 3-A, the PAO activity increased, and reached the highest level at 6 h, then decreased gradually, but it was still significantly higher than that of the control and maintained at a high level.
Fig. 3 Effects of Cd on the activity of PAO and the content of H2O2 in roots of PYTC seedlings. A, the activity of PAO. B, the content of H2O2 .
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
JIANG Qian-qian et al.
1132
tures were kept in normal morphology. The uniform nucleoplasm and clear nucleolus could be observed (Fig. 5-A). After 200 µmol L-1 Cd treatment for 48 h, condensation of chromatin, deformation of nucleus, shrinkage or invagination of nuclear membrane were observed in the nucleus of PYTC extensive roots. The boundaries of nuclear membrane blurred and some parts broken. So the nucleoplasm leaked into the cytoplast. Finally, the nuclei disappeared and the chromatin condensed into a half-moon form, located on the edge (Fig. 5-B and C). As shown in Fig. 5-D, the mitochondrial morphology did not appear abnormal: the mitochondrial structure was clear, the inner and outer membrane and the cristae were all integrated in the extensive roots of PYTC seedlings without Cd stress. After 200 µmol L -1 Cd treatment, mitochondria were distorted and swollen, the structure of outer membrane and cristae became blurred, and the cristae was gradually broken and dissolved, small vacuolation appeared in mitochondria, and the vacuolation expanded until the disintegration of some mitochondria (Fig. 5-E and F). The structural changes in nucleus and mitochondria indicated the occurrence of PCD in roots of PYTC under Cd stress. Fig. 4 Effects of Cd on the cell death in roots of PYTC seedlings. A, treatment of different concentrations Cd for 48 h. B, treatment of 200 µmol L-1 Cd for 0 (control), 0.5, 1, 2, 4, 6, 12, 24, 48, 72, and 96 h.
centration was over 200 µmol L -1. These results showed that, after 48 h treatment, 200 µmol L-1 Cd could increase the membrane permeability then resulted in substantial cell death. However, Fig. 4-B showed that 200 µmol L-1 Cd had no obvious effect on the cell death in the early state of treatment (
