Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis thaliana cell suspensions

with the design of the microarray experiments and Radek Zíka (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Czech Republic) for help with the promoter analysis evaluation. We are grateful to Dr. Plant Science Center, Sweden) for careful reading of the manuscript. 4 Abstract (223 words): Salicylic acid (SA) plays a central role in defense against pathogen attack, as well as in germination, flowering, senescence and the acquisition of thermotolerance. In this report we investigate the involvement of phospholipase D (PLD) in the SA signaling pathway. In presence of exogenous primary alcohols, the production of phosphatidic acid (PA) by PLD is diverted towards the formation of phosphatidylalcohols through a reaction called transphosphatidylation. By in vivo metabolic phospholipid labeling with 33 P i , PLD activity was found to be induced 45 min after addition of SA. We show that incubation of Arabidopsis thaliana cell suspensions with primary alcohols inhibited the induction of two SA-responsive genes, PR1 and WRKY38, in a dose dependent manner. This inhibitory effect was more pronounced when the primary alcohols were more hydrophobic. Secondary or tertiary alcohols had no inhibitory effect. These results provide compelling arguments for PLD activity being upstream of the induction of these genes by SA. A subsequent study of n-butanol effects on the SA responsive transcriptome identified 1327 genes differentially expressed upon SA treatment. Strikingly, the SA-response of 380 of these genes was inhibited by n-butanol but not by tert-butanol. A detailed analysis of the regulation of these genes showed that PLD could act both positively and negatively, either on gene induction or gene repression. The overlap with the previously described Phosphatidylinositol-4-Kinase pathway is discussed.