scholarly journals Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

2005 ◽  
Vol 389 (3) ◽  
pp. 695-704 ◽  
Author(s):  
Sandra Matic ◽  
Daniela A. Geisler ◽  
Ian M. Møller ◽  
Susanne Widell ◽  
Allan G. Rasmusson

The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)+. After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD+). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaceous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed.

2021 ◽  
Vol 22 (24) ◽  
pp. 13368
Author(s):  
Agnieszka Kobylińska ◽  
Małgorzata Maria Posmyk

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin effectively eliminates oxidative stress (direct and indirect antioxidant) and switches on different defence strategies (preventive and interventive actions) during environmental stresses. In the presented report, exogenous melatonin potential to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) exposed to lead against death was examined. Analyses of cell proliferation and viability, the level of intracellular calcium, changes in mitochondrial membrane potential (ΔΨm) as well as possible translocation of cytochrome c from mitochondria to cytosol and subsequent caspase-like proteolytic activity were conducted. Our results indicate that pretreatment BY-2 with melatonin protected tobacco cells against mitochondrial dysfunction and caspase-like activation caused by lead. The findings suggest the possible role of this indoleamine in the molecular mechanism of mitochondria, safeguarding against potential collapse and cytochrome c release. Thus, it seems that applied melatonin acted as an effective factor, promoting survival and increasing plant tolerance to lead.


2017 ◽  
Vol 474 (10) ◽  
pp. 1689-1703 ◽  
Author(s):  
Frédéric Toussaint ◽  
Baptiste Pierman ◽  
Aurélie Bertin ◽  
Daniel Lévy ◽  
Marc Boutry

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia, which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min−1 mg−1) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters.


PROTOPLASMA ◽  
2003 ◽  
Vol 221 (1-2) ◽  
pp. 93-100 ◽  
Author(s):  
T. Raeymaekers ◽  
G. Potters ◽  
H. Asard ◽  
Y. Guisez ◽  
N. Horemans

2018 ◽  
Vol 5 (10) ◽  
pp. 2269-2281 ◽  
Author(s):  
Yanhui Dai ◽  
Zhenyu Wang ◽  
Jian Zhao ◽  
Lili Xu ◽  
Lina Xu ◽  
...  

This study investigated the toxicity of CuO nanoparticles (NPs) to Nicotiana tabacum L. cv. Bright Yellow-2 (BY-2) cells.


2021 ◽  
Vol 12 ◽  
Author(s):  
Xavier Herman ◽  
Johann Far ◽  
Adeline Courtoy ◽  
Laurent Bouhon ◽  
Loïc Quinton ◽  
...  

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.


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