Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Czech Republic

Institute of Experimental Botany of the Czech Academy of Sciences Czech Republic

Institute of Experimental Botany of the Czech Academy of Sciences Czech Republic Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Czech Republic

Plant Protection Institute Centre for Agricultural Research Hungarian Academy of Sciences Hungary

The Institute of Bioorganic Chemistry and Petrochemistry National Academy of Sciences of Ukraine Ukraine

References provided by Crossref.org