Interaction Between Phosphatidic Acid And MPK6/MKK9in Response To Salt Stress In Arabidopsis Thaliana

Phospholipase D (PLD) hydrolyzes phospholipids to produce phosphatidic acid (PA) and a free head group, such as choline, ethanolamine, or serine. Twelve PLD genes are present in the genome of the model plant (Arabidopsis thaliana). PLD has been reported to be involved in many signal transduction processes in plant cell. For example abscisic acid (ABA), freezing, wounding, dehydration, pathogen invasion and salt stress. PA is believed to act as an impotrant signal molecule. It is generated rapidly and transiently during various stress responses. PA mediates the growth and development of plant through the interaction with taget proteins.MAPK (mitogen-activated protein kinase) cascades are conversed signalling modules found in all eukaryotic cells including plant, fungi and animals. A MAPK cascade consists of a MAPKKK-MAPKK-MAPK module that links from receptors to downstream targets. They transmit extracellular stimuli into intracellular responses while amplifying the transducing signal. MAPK cascades play a remarkably important role in plant signalling of a variety of abiotic and biotic stresses.In the previous study of our lab it found that PA stimulated the MPK6kinase activity.According to these results, to understand the relation between PA and MPK6, MPK6was immunoprecipitated from leaf protein extracts of WT with an MPK6-specific antibody. When16:0-18:2PA was incubated with immunoprecipitated MPK6protein, it stimulated the kinase activity.16:0-18:2PC at the same concentrations with PA showed no stimulatory effect on the MPK6kinase activity. We compared MPK6kinase activity in WT and pldal seedlings. Endogenous kinase activity of MPK6from pldal and WT leaf extracts was determined after NaCl treatment using immunocomplex kinase assays. NaCl treatment resulted in a rapid activation of endogenous MPK6in WT, but not in pldal mutant. Protein levels of MPK6kept constant through western blotting with an MPK6-specific antibody during the NaCl treatment. These data suggested that PLDα1-derived PA bound to MPK6and leaded to the kinase activation in response to NaCl stress, and the activation of MPK6was mainly induced by posttranslational modification.Genetic studies showed the interaction between PA and MPK6with homozygous pldal/mpk6double mutant lines generated by genetic crosses. Seeds of the wild type pldal, mpk6, and pldal/mpk6double mutant were grown on1/2MS plates with or without NaCl. After6days, gemination rate of pldal/mpk6was lower than pldal mutant and mpk6mutant. Furtermore, seedlings of WT, pldal, mpk6and pldal/mpk6grown on MS plates for4days were tansferred to1/2MS plates with or without NaCl. After8days primary root growth of pldal, mpk6and pldal/mpk6null mutants were inhibited, and degree of inhibition increased in turn. Seedlings of pldallmpk6on NaCl medium showed complete chrolosis. These results suggested PLD, PA and MPK6responded to and mediated salt stress tolerance in plantsIn Arabidopsis SOS1(salt overly sensitive1) is an important Na+/H+antiporter. In the pull down assay MPK6directly interacted with the soluble parts of SOS1in vitro. Six SOS1cDNA fragments for C-terminal were deleted. Furthermore, MPK6bound to SOS1CT7(1044to1146amino acids) through pull-down assay. MPK6immunoprecipitated from the plants could phosphorylate GST-SOS1CT7. Futhermore, the phosphorylation was more efficient when MPK6was inmmunoprecipitated forn the plants treated with NaCl. PA increased the effect on MPK6phosphorylation of SOS1CT7.On the other hand, Five MKKs genes, MKK1, MKK2, MKK5, MKK6and MKK9cloned according to Arabidopsis genome were linked to GST-tag vector and expressed in Escherichia coli. It was found that only MKK9strongly bound to PA and didn’t bind to other lipids through protein-lipid nitrocellulose filter-binding assay. MKK9protein expressed and purified from E.coli phosphorylated MPK6-HIS fusion protein. Applied exogenous PA (16:0-18:2) increased the activity of phosphorylation of MPK6by MKK9. Single constitutively active MKK9(EE)-Flag protein or constitutively active MKK9(EE)-Flag and MPK6-HA proteins were transiently expressed in Arabidopsis protoplasts. Treatment with PA stimulated MKK9activity to MPK6-GST and MPK6activity to MBP (myelin basic protein). Interestingly NaCl treatment inhibited the activity of phosphorylation of inactive MPK6(Km)-GST by constitutively active MKK9(EE)-Flag in protoplasts, but increased MPK6activity to MBP when MKK(EE)-Flag and MPK6-HA were coexpressed. The results suggested the PA bound to MPK6and MKK9simultaneously and simulated the activity of phosphorylation of MPK6by MKK9.