title: A novel mechanism of aluminum-induced cell death involving vacuolar processing enzyme and vacuolar collapse in tobacco cell line BY-2.
abstract: The role of vacuole in the cell death mechanism induced by aluminum (Al) was investigated in tobacco (Nicotiana tabacum L.) cell line BY-2. Cells at logarithmic phase of growth were treated without (control) or with Al (up to 150 μM) in a treatment medium containing CaCl2, sucrose and 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.0). After 18 h treatment, both the integrity of the plasma membrane (estimated by Evans blue uptake) and growth capacity (estimated by post-Al treatment growth in nutrient medium) were decreased, while the activity of vacuolar processing enzyme (VPE) was increased, in the Al dose-dependent manner. The activity of the vacuole (estimated by neutral red uptake) was slightly increased at 50 μM then decreased with an increase in Al concentration. Direct observation of morphological changes of vacuole in a transgenic BY-2 expressing GFP-AtVam3p fusion protein localized on tonoplast indicated Al-induced collapse of vacuole. Time-course experiments indicated that both an increase in VPE activity and a loss of growth capacity were clearly observed at 6 h of the treatment time, prior to the loss of plasma membrane integrity. The presence of Ac-YVAD-CHO (an inhibitor effective to VPE) during Al treatment suppressed a loss of plasma membrane integrity. The expression of VPE genes (VPE-1a, VPE-1b) were significantly enhanced by Al treatment. Taken together, we conclude that an enhancement of VPE activity by Al is controlled at transcriptional level, and is a key factor leading to a loss of integrity of the plasma membrane and a loss of growth capacity.
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Kariya
authors.forename: Tijen
authors.initials: T
authors.lastname: Demiral
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sasaki
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Ismail
authors.initials: I
authors.lastname: Turkan
authors.forename: Toshio
authors.initials: T
authors.lastname: Sano
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Hasezawa
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamamoto
mesh_terms: D000535:Aluminum / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D010940:Plant Proteins; C096429:L 709049; D049452:Green Fluorescent Proteins; D000535:Aluminum; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme
keywords: Aluminium toxicity; Cell death; Evans blue; Neutral red; Vacuolar processing enzyme (VPE)