Paula Teper-Bamnolker _Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel.
Raz Danieli _ Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel; Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem , Rehovot 76100, Israel.
Hadas Peled-Zehavi _ Department of Plant and Environmental Sciences, The Weizmann Institute of Science , Rehovot 76100, Israel.
Eduard Belausov _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Mohamad Abu-Abied _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Tamar Avin-Wittenberg _ Department of Plant and Environmental Sciences, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram , Jerusalem, 9190401, Israel.
Einat Sadot _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Dani Eshel _ Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel.
The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.
Paula Teper-Bamnolker _Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel.
Raz Danieli _ Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel; Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem , Rehovot 76100, Israel.
Hadas Peled-Zehavi _ Department of Plant and Environmental Sciences, The Weizmann Institute of Science , Rehovot 76100, Israel.
Eduard Belausov _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Mohamad Abu-Abied _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Tamar Avin-Wittenberg _ Department of Plant and Environmental Sciences, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram , Jerusalem, 9190401, Israel.
Einat Sadot _ Department of Ornamental Horticulture, The Volcani Center, ARO , Rishon LeZion, Israel.
Dani Eshel _ Department of Postharvest and Food Sciences, The Volcani Center, ARO , Rishon LeZion, Israel.
The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.