Involvement of protein kinases and calcium in the NO-signalling cascade for defence-gene induction in ozonated tobacco plants

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O3) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca2+ in defence-gene activation. NO was visualised with the NO probe DAF-FM. Using a pharmacological approach, we monitored the effects of different inhibitors on the expression profiles of NO-dependent defence genes, using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O3 stimulates a 48-kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O3-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. In contrast, protein dephosphorylation mediates pathogenesis related protein 1 a (PR1a) expression in O3-treated tobacco plants. Ca2+ is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca2+ transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca2+ suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA and the cellular messengers in this O3-induced defence gene activation is proposed.