Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLD alpha 1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H2O2) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLD alpha 1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO4. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLD alpha 1-deficient cells. Compared to WT, in CPT-treated PLD alpha 1 mutant cells, reduced cell death and decreased production of H2O2 were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLD alpha 1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLD alpha 1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLD alpha 1 is an important factor in chemical-induced PCD signal transduction