Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts

Among plant defense responses to pathogen attack, the release of active oxy gen species (AOS), termed the oxidative burst, may affect the attacking pat hogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxi dase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protopl asts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsi s protein phosphatase 2A abolished this enhancement, whereas Arabidopsis du al-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect. tMEK2(MUT), a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity w hen overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2(MUT), also enhanced production of AOS in Intact protoplasts. Our results show that ec topic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.