| The phospholipid signaling pathway in plants plays important roles in the growth and response to environmental stress as phospholipases are key enzymes catalyzing phospholipids and generating second messengers.Phospholipases are divided into three classes,phospholipase A(PLA),PLC and PLD according to their different hydrolysis sites.Two classes of PLCs have been identified in plants according to the substrates they catalyze:the nonspecific PLCs(NPC)and phosphoinositide-specific PLCs(PI-PLC).There are four PI-PLCs had been detected in rice,however,the mechanism of OsPLC is still unclear.In this study,we use wild-type rice and Tosl7 insertion OsPLC4 mutant as materials to investigate OsPLC1 roles in salt response.We focused on cellular localization OsPLC1 and its selectivity for substrates.We constructed the ProPLC1::GFP-OsPLC1 vector,transformed it to tobacco leaves by agrobacterium and observed by confocal microscope.The result suggests that ProPLC1::GFP-OsPLC1 localizes in plasma membrane and cytoplasm.Subsequently,we transformed pSuper::GFP-OsPLC1 and ProPLC1::GFP-OsPLC1 into WT rice and obtained the transgenic plants with pSuper::GFP-OsPLC1.The intracellular distribution of OsPLC1 in leaves was observed by confocal microscope.The pSuper::GFP-OsPLC1 was localized with both plasma membrane and cytoplasm.In order to further determine the location of OsPLC1,we used two phase method to separate the plasma membrane and cytoplasm,and then use the GFP antibody for Western Blotting.The results showed that GFP-OsPLC1 localized in plasma membrane and cytoplasm.GFP-OsPLC1 in plasma membrane deceases and GFP-OsPLC1 in cytoplasm increases after salt treatment.So we predict that OsPLC1 is in cytoplasm under normal condition,and recruited partially to plasma membrane under NaC1 stimulation.Molecular mass of GFP-OsPLC1 in plasma membrane was larger than that in cytoplasm.This change in molecular mass of OsPLC1 is probably owing to protein modification.We also screened for proteins interacting with OsPLC1 by yeast two-hybrid system,identifying JOKA2 as a putative partner.In animal cells,PLC mainly hydrolyzes phosphatidylinositol 4,5-bisphosphate(PtdIns(4,5)P2).However,the content of phosphatidylinositol 4-phosphate(PtdIns4P)is much higher than that of PtdIns(4,5)P2 in plants.We carried out studies to determine OsPLC1 activity towards to PtdIns4P and PtdIns(4,5)P2.Firstly we cloned specific-binding domains PHFAPP1 and PHPLC?1 of Ptdlns4P and PtdIns(4,5)P2,and then constructed their GFP fusioned expression vector to probe PtdIns4P and PtdIns(4,5)P2 in vivo.Results showed that PtdIns(4,5)P2 and PtdIns4P are mainly localized in plasma membrane and PtdIns4P shows polar distribution in rice roots.PHPLC?1-GFP localized in cytoplasm besides in plasma membrane,suggesting low content of PtdIns(4,5)P2 in rice thus some PHPLC?1-GFP can not be bound to PtdIns(4,5)P2 on plasma membrane.We next used the PHFAPP1-GFP transgenic plants to detect the changes of PtdIns4P content after 75 mM NaCl treatment.The results showed that the content of PtdIns4P decreased significantly after salt treatment.Then we used thin layer chromatography and gas chromatography to determine the content of PtdIns4P in the wild type and the osplc1 mutant before and after 150 mM NaCl treatments.The changes of PtdIns4P content were consistent with those determines by the biosensors.Therefore,we hypothesized that the reduction of PtdIns4P content in the wild type was due to the hydrolysis of OsPLC1 after the salt treatment.However,due to PtdIns(4,5)P2 content in plants is very low,these two methods were unable to detect the content of PtdIns(4,5)P2.Taken together,we used methods of molecular biology,cell biology,biochemistry to determine the cellular localization of OsPLC1,PtdIns(4,5)P2 and PtdIns4P.According to the detection of PtdIns4P content in the wild type and the mutant before and after salt treatment,we preliminarily demonstrate that OsPLC1 can hydrolysis PtdIns4P under salt stress. |
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