A novel mechanism of aluminium-induced cell death involving vacuolar processing enzyme and vacuolar collapse in tobacco cell line BY-2

•Vacuolar processing enzyme (VPE) is known to be a cell death executioner.•Aluminum ion increases VPE activity and vacuolar collapse in cultured tobacco cells.•An increase in VPE activity by Al is controlled transcriptionally.•Vacuole is involved in Al-induced cell death process in plant cells.

The role of vacuole in the cell death mechanism induced by aluminium (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.

Graphical abstractA novel mechanism of aluminium-induced cell death involving vacuolar processing enzyme and vacuolar collapse in BY-2 tobacco cells.Figure optionsDownload full-size imageDownload as PowerPoint slide