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Live-Cell Imaging of Dual-Labeled Golgi Stacks in tobacco BY-2 cells Reveals Similar Behaviors for Different Cisternae during Movement and Brefeldin A Treatment

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Molecular Plant 2011年第5期4卷 896-908页

作者： Stephanie L. Madison Andreas Nebenfuhr Department of Biochemistry and Cellular and Molecular Biology University of Tennessee Knoxville TN 37996-0840 USA

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.

关键词： Golgi apparatus Golgi stack integrity brefeldin A monensin tobacco BY-2 cells live-cell imaging.

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Dihydrosphingosine-lnduced Programmed Cell Death in tobacco BY-2 cells Is Independent of H2O2 Production

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Molecular Plant 2011年第2期4卷 310-318页

作者： Christophe Lachaud Universite de Toulouse UPS UMR 5546 Surfaces Cellulaires et Signalisation chez les Vegetaux BP 42617 F-31326 Castanet-Tolosan France CNRS UMR 5546 BP 42617 F-31326 Castanet-Tolosan France

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.

关键词： tobacco BY-2 cells calcium signaling cytosolic calcium aequorin sphingolipids LCBs dihydrosphingosine sphinganine apoptosis Programmed Cell Death （PCD） Reactive Oxygen Species （ROS） H2O2 oxidative burst.

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Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

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Journal of Integrative Plant Biology 2015年第1期57卷 127-135页

作者： Yumi Hirakawa Toshihisa Nomura Seiichiro Hasezawa Takumi Higaki Department of Integrated Biosciences Graduate School of Frontier Sciences The University of Tokyo RIKEN Center for Sustainable Resource Science

Vacuoles are suggested to play crucial roles in plant defense-related cell death. During programmed cell death, previous live cell imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole＇s rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cell cycle-synchronized tobacco BY-2 （Nicotiana tabacum L. cv. Bright Yellow 2） cells during ceil death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90% of the cells by 24 h. Prior to cell death, vacuole shape simplified and endoplasmic actin filaments disassembled; however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cell death.

关键词： Actin filaments defense-related cell death Erwiniacarotovora tobacco BY-2 cells vacuoles

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Ectopic Expression of a Bacterium NhaD-type Na^+/H^+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

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Journal of Integrative Plant Biology 2012年第6期54卷 412-421页

作者： Nai-Qin Zhong Li-Bo Han Xiao-Min Wu Li-Li Wang Fang Wang Yan-He Ma Gui-Xian Xia Institute of Microbiology Chinese Academy of SciencesBeijing 100101 State Key Laboratory of Plant Genomics Beijing 100101

AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

关键词： Alkaliphiles Alkalimonas amylolytica Na＋/H＋ antiporter tobacco BY-2 cells high salinity stress alkaline.

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