The Roles Of PDK1 S-Nitrosylation In No-Induced Cell Death In Plants

It has been known for over a decade that NO can trigger cell death in plants. However, the molecular mechanism behind the NO-triggered cell death is not understood. We observed that silencing GSNOR1 (S-nitrosoglutathione reductase), encoding a key enzyme that regulates cellular levels of S-nitrosylation, resulted in significant spontaneous cell death on the leaves of silenced tomato, N tabacum and N. benthamiana plants, respectively. TMV infection could trigger significant systemic cell death on GSNOR1-silenced N. benthamina plants. Given the significantly enhanced level of protein S-nitrosylation in the GSNOR1-silenced plants, we postulated that the spontaneous cell death on GSNOR1-silenced plants could be resulted from S-nitrosylation and thus the inhibition of a key negative regulator of cell death. Based on the current literature, we targeted on PDK1-PKB/Akt/Adi3 pathway, which has been implicated in negatively regulating cell death in yeast, mice and tomato as PKB/Akt, the tomato Adi3 homolog, in mice has been shown as a target of S-nitrosylation and its kinase activity is inhibited by this modification. To test whether the tomato Adi3 is similarly subjected to S-nitrosylation, biotin switch assay was performed. To our surprise, PDK1, the AGC kinase functions upstream of Adi3, but not Adi3 was S-nitrosylated in the presence of GSNO. It has been reported previously that silencing PDK1 in tomato caused lethal phenotype. The kinase activity of PDK1 was inhibited by GSNO in vitro in a concentration-dependent manner, indicating that PDK1 activity is regulated by S-nitrosylation. This inhibition was reversible by reducing agent DTT but synergistically enhanced by H2O2. Four Cysâ†'Ser mutants of PDK1 have been generated by in vitro mutagenesis and these mutants will help to identify the target Cys for S-nitrosylation. Taken together, our results might help to establish a potential link between NO-triggered cell death and PDK1, a conserved cell death regulator in yeast, mamma and plants.