Phosphatidic Acid Regulates MKK7 And MKK9 In Arabidopsis Thaliana In Respons To Salt Stress

MAPK (mitogen-activated protein kinase) cascade is a highly conserved signaling pathway among eukaryotes. MPK signaling cascades are composed of three main signaling elements. In a general model, stimulated plasma membrane receptors activate MAP kinase kinase kinases, sequential phosphorylations ensue as MAPKKKs activate downstream MAP kinase kinases that in turn activate MAPKs. MAPKs then target various effector proteins in the cytoplasm or nucleus, which include other kinases, enzymes, or transcription factors. There are 20 MAPKs,10 MAPKKs, and more than 60 MAPKKKs in the Arabidopsis genome based on sequence homology. The MAPK cascade has been shown to be involved in various biotic and abiotic stress responses, hormone responses, cell proliferation, differentiation, and developmental processes in plants.Phospholipid metabolism plays an important role in various signal transduction pathways in both higher plants and animals. In stress-induced signal transduction, phosphatidic acid (PA) responses have been mainly attributed to two pathways. It is the direct product of phospholipase D (PLD), which hydrolyses structural phosholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and a secondary product of the phospholipase C (PLC) pathway, which first hydrolyzes polyphosphoinositides (PPIs) to diacylglycerol (DAG), that is subsequently phosphorylated to PA by diacylglycerol kinase (DGK). PA functions as a second messenger to regulate protein kinases, protein phosphatase, small G proteins and proteins involved in cytoskeletal rearrangements in response to cellular processes and plant physiological responses.The previous study of our lab found out that PA regulated MPK6 in response to salt stress. In this case, we proposed a hypothesis that whether there was an upstream kinase involved in this signaling pathway. It had been indicated in our previous work that PA was not interacted with MKK1, MKK2, MKK5 and MKK6. In this work, we expressed and purified His tagged MKK7 and MKK9 in Escherichia coli. By protein-lipid binding assay, we found that both MKK7 and MKK9 were interacted with PA strongly, and identified which kinds of PA species could interact with them. It had been reported that MKK9 is an upstream kinase of MPK6. So we decided to study whether MKK7 is also an upstream of MPK6. By in vitro kinase assay and gel mobile shift assay, we proved that MKK7 phosphorylated MPK6 both in vitro and in vivo. Based on that, we studied the influence of PA on the activation of MKK7 and MKK9. First of all, we transiently expressed Flag tagged MKK7 and MKK9 in Arabidopsis protoplasts. The protoplasts were treated by 25 mM NaCl and PA, and measured the activity of MKK7 and MKK9 by in vitro kinase assay used MPK6(Km) as a substrate. As a consequence, the activity of MKK7 and MKK9 was increased after both two kinds of treatments. Besides, when we perform the same experiment in pldal protoplasts the activity of MKK7 and MKK9 was not increased as that in WT protoplasts. This result demonstrated that PLDal null block the relationship between salt signal and MKK7/MKK9. The activation of MKK7 and MKK9 was resulted from the generation of PA by PLDal after salt treatment. PLDal/PA is located in the upstream of MAPK cascade in this signal pathway.In MAPK cascade, the activated MKKs phosphorylate and activate the downstream MPKs. We tested whether MKK7 and MKK9 activated by salt treatment could activate MPK6. We treated the seedlings of WT, mkk7, mkk9 and mkk7/9 with 100 mM NaCl and measured the phosphorylation of MBP as s substrate. Different to the WT seedlings, activity of MPK6 in mutant seedlings was not increased obviously. This result showed that loss of function of MKK7 and MKK9 disabled the activation of MPK6 in response to salt stress. When we expressed MPK6 with MKK7 or MKK9 in protoplasts, the activity of MPK6 was much higher than that was expressed alone. Above all, we confirmed that MKK7 and MKK9 were two positive regulators in mediation of MPK6 in response to salt stress.In the previous work in our lab, MPK6 could phosphorylate SOS1, a Na+/H+ transporter located on plasma membrane, to promote the exclusion of Na+ in response to salt stress. To find out the mechanism associated MAPK cascade with SOS1 in space, we contrasted the content of MKK7/MKK9 by immunoblotting assay before and after PA treatment for 15 min. We found that in contrast with that in cytoplasm, the amount of MKK7/MKK9 on plasma membrane was increased. The protoplasts isolated from WT and pldal were transformed by MKK7/MKK9 and treated by NaCl for 60 min. We found that the effect of NaCl treatment on spatial distribution of MKK7/MKK9 in WT protoplasts was same as that of PA treatment. Besides, there were no distribution changes in pldα1 protoplasts. Above all, we summarized the conclusion that the distribution changes of MKK7/MKK9 upon salt stress were resulted in the effect of PA which is generated by PLDal. We further tested this conclusion in the respect of cellular biology, MKK7 and MKK9 were generally colocalization with MPK6 on the plasma membrane, in the cytoplasm and in the nucleus of the protoplasts. When the protoplasts were exposure to 20 μM PA, we could observe the movement of MKK7/MKK9 from the cytoplasm to the plasma membrane in protoplasts by Laser Scanning Confocal Microscope. Same as the distrbution changes analyzed by immunoblotting assay, this certain kind of movement was observed in WT protoplasts but not in pldα1 protoplasts after treated by NaCl for 15-20 min. This result suggested that PA acted as a scaffold on the spatial distribution of MKK7/MKK9 in cellular upon salt stress.