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.

• Klíčová slova
• Arabidopsis thaliana, MAMP-triggered immunity, Pseudomonas syringae, effector-triggered immunity, flagellin, non-specific phospholipase C, phosphatidylcholine-specific phospholipase C, reactive oxygen species,
• MeSH
• Arabidopsis enzymologie   imunologie   mikrobiologie   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfolipasy typu C genetika   fyziologie   MeSH
• Golgiho aparát enzymologie   MeSH
• imunita rostlin fyziologie   MeSH
• klonování DNA MeSH
• konfokální mikroskopie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• nemoci rostlin imunologie   mikrobiologie   MeSH
• proteiny huseníčku genetika   fyziologie   MeSH
• protoplasty enzymologie   MeSH
• Pseudomonas syringae * MeSH
• reaktivní formy kyslíku MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylcholiny MeSH
• fosfolipasy typu C MeSH
• NPC2 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• reaktivní formy kyslíku MeSH

BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

• MeSH
• endocytóza MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• pyridinové sloučeniny metabolismus   MeSH
• rostlinné buňky fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   fyziologie   MeSH
• transport proteinů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• FM 4-64 MeSH Prohlížeč
• kvartérní amoniové sloučeniny MeSH
• pyridinové sloučeniny MeSH
• rostlinné proteiny MeSH
• výměnné faktory guaninnukleotidů MeSH

The Arabidopsis non-specific phospholipase C (NPC) protein family is encoded by the genes NPC1 - NPC6. It has been shown that NPC4 and NPC5 possess phospholipase C activity; NPC3 has lysophosphatidic acid phosphatase activity. NPC3, 4 and 5 play roles in the responses to hormones and abiotic stresses. NPC1, 2 and 6 has not been studied functionally yet. We found that Arabidopsis NPC1 expressed in Escherichia coli possesses phospholipase C activity in vitro. This protein was able to hydrolyse phosphatidylcholine to diacylglycerol. NPC1-green fluorescent protein was localized to secretory pathway compartments in Arabidopsis roots. In the knock out T-DNA insertion line NPC1 (npc1) basal thermotolerance was impaired compared with wild-type (WT); npc1 exhibited significant decreases in survival rate and chlorophyll content at the seventh day after heat stress (HS). Conversely, plants overexpressing NPC1 (NPC1-OE) were more resistant to HS compared with WT. These findings suggest that NPC1 is involved in the plant response to heat.

• Klíčová slova
• Arabidopsis thaliana, diacylglycerol, heat stress, non-specific phospholipase C, phospholipids,
• Publikační typ
• časopisecké články MeSH