A rapid and concentration-dependent generation of superoxide anion （·O2^-）, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts （LaC...A rapid and concentration-dependent generation of superoxide anion （·O2^-）, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts （LaCl3 and CdCl3 ） were added to tobacco （ Nicotiana tabacum） cell suspension culture. Addition of superoxide dismutase （480 U·ml^-1） and Tiron （5 μmol·L^-1） to cell culture suspension decreases the level of lanthanide cation-induced ·O2^- generation, suggesting that ·O2^- generation is extra-cellular. Pretreatment of the cell culture suspension with diphenyleneiodonium （10 and 50 μmol·L^-1 ）, quinacrine （ 1 and 5 mmol· L^-1 ） and imidazol （ 10 mmol· L^-1 ）, inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase. In addition, addition of SHAM （1 and 5 mmol· L^-1）, azide （0.2 and 1 mmol· L^-1 ）, inhibitor of peroxidase, has no influence on ·O2^- generation.展开更多
Microcystin-RR （MC-RR） has been suggested to induce apoptosis in tobacco BY-2 cells through mitochondrial dysfunction including the loss of mitochondrial membrane potential . TO further elucidate the mechanisms invo...Microcystin-RR （MC-RR） has been suggested to induce apoptosis in tobacco BY-2 cells through mitochondrial dysfunction including the loss of mitochondrial membrane potential . TO further elucidate the mechanisms involved in MC-RR induced apoptosis in tobacco BY-2 cells, we have investigated the role of mitochondrial electron transport chain （ETC） as a potential source for reactive oxygen species （ROS）. Tobacco BY-2 cells after exposure to MC-RR （60 mg/L） displayed apoptotic changes in association with an increased production of ROS and loss of Am. All of these adverse effects were significantly attenuated by ETC inhibitors including Rotenone （2 μmol/L, complex I inhibitor） and antimycin A （0.01 μmol/L, complex III inhibitor）, but not by thenoyltrifluoroacetone （S μmol/L, complex Ⅱinhibitor）. These results suggest that rnitochondrial ETC plays a key role in mediating MC-RR induced apoptosis in tobacco BY-2 cells through an increased mitochondrial production of ROS.展开更多
Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases an...Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.展开更多
In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusua...In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A （BFA）-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a re- markable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addi- tion, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.展开更多
To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cellattached membrane capacitance measurements. These measurements revealed different modes of fusion and fission...To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cellattached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events, fusion occurred transiently, which facilitates rapid recycling of vesicles. In addition, transient sequential or multi-vesicular exocytosis observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together, the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.展开更多
文摘A rapid and concentration-dependent generation of superoxide anion （·O2^-）, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts （LaCl3 and CdCl3 ） were added to tobacco （ Nicotiana tabacum） cell suspension culture. Addition of superoxide dismutase （480 U·ml^-1） and Tiron （5 μmol·L^-1） to cell culture suspension decreases the level of lanthanide cation-induced ·O2^- generation, suggesting that ·O2^- generation is extra-cellular. Pretreatment of the cell culture suspension with diphenyleneiodonium （10 and 50 μmol·L^-1 ）, quinacrine （ 1 and 5 mmol· L^-1 ） and imidazol （ 10 mmol· L^-1 ）, inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase. In addition, addition of SHAM （1 and 5 mmol· L^-1）, azide （0.2 and 1 mmol· L^-1 ）, inhibitor of peroxidase, has no influence on ·O2^- generation.
基金supported by the National Natural Science Foundation of China (No.31100340)the Major Science and Technology Program for Water Pollution Control and Treatment (No.2012ZX07103-004-02)
文摘Microcystin-RR （MC-RR） has been suggested to induce apoptosis in tobacco BY-2 cells through mitochondrial dysfunction including the loss of mitochondrial membrane potential . TO further elucidate the mechanisms involved in MC-RR induced apoptosis in tobacco BY-2 cells, we have investigated the role of mitochondrial electron transport chain （ETC） as a potential source for reactive oxygen species （ROS）. Tobacco BY-2 cells after exposure to MC-RR （60 mg/L） displayed apoptotic changes in association with an increased production of ROS and loss of Am. All of these adverse effects were significantly attenuated by ETC inhibitors including Rotenone （2 μmol/L, complex I inhibitor） and antimycin A （0.01 μmol/L, complex III inhibitor）, but not by thenoyltrifluoroacetone （S μmol/L, complex Ⅱinhibitor）. These results suggest that rnitochondrial ETC plays a key role in mediating MC-RR induced apoptosis in tobacco BY-2 cells through an increased mitochondrial production of ROS.
文摘Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.
文摘In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A （BFA）-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a re- markable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addi- tion, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.
文摘To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cellattached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events, fusion occurred transiently, which facilitates rapid recycling of vesicles. In addition, transient sequential or multi-vesicular exocytosis observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together, the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.
