Molecular Mechanisms Of Phospholipase D In Regulating Auxin Signal Transduction In Response To Salt Stress In Arabidopsis

Soil salinity is a global ecological problem,which has a great negative impact on ecological environment and agricultural production.Salt stress causes plant growth and development retardation,leading to many physiological reactions.The formation of plant physiology,cellular and genetic mechanism to survive in the high salt stress include SOS(Salt overly sensitive)system,plant hormone,antioxidant defense system,osmolytes and lipids signal etc.Phospholipase D(PLD)is a key enzyme that catalyzes lipids in response to abiotic stresses.PLD and its production phosphatidic acid(PA)participate in plant response to various stresses.Therefore,we investigate the molecular mechanism of PLD and PA in regulating plant salt tolerance,which is essential for the study of plant salt resistance and crop genetic enhancement.Arabidopsis genomes have 12 PLD genes,which are divided into PLDa(3),PLD?(2),PLD?(3),PLD?,PLD? and PLDC(2)according to the protein structure and catalytic region.Among them,PLDa1 and PLD? are the most abundant.In this study,the model plant Arabidopsis thaliana was used as experimental materials to study the regulation of PLD and PA on plant kinase protein PID activity and its effect on salt tolerance.The main results are as follows:Western blotting were performed to verify the pld mutant materials including pldal,pld?-1,pld?-2,plda1 pld?-1 and plda1 pld?-2.PLDa and PLDS activity was measured in wild type(WT)and pld mutants using 3H-PC(phosphatidylcholine)and specific enzyme reaction buffer solution.The results showed that the PLD activity in pld single mutant was significantly lower than that in the WT,while the activity of PLD activity in plda1 pld?-1 and plda1 pld?-2 was the lowest.After 3 min treatment with 125 mM NaCl,the PLD total activity(PLDa and PLD?)increased significantly,maximized at 5 min,followed by a decrease after 30 min of NaCl treatment.Meanwhile,we determined the phospholipid components of Arabidopsis thaliana by ESI-MS/MS.The results show that,after 10 min treatment,the total PA content of WT increased significantly,mainly including molecular species 34:2 PA,34:3 PA,36:4 PA,36:5 PA,and 36:6 PA,but there was no significant change in plda1 pld?-1.Treatment of WT and pld mutant seedlings with 125 mM NaCl,7 days after the taproot length statistics found that NaCl significantly inhibited primary root elongation of WT and pld single mutants,and greater inhibition was found in plda1 pld?-1 and plda1 pld?-2.Exogenous application of PA restored primary root growth in the pld mutant to WT levels partially when exposed to NaCl.These results suggest that PLD and its product PA are involved in salt tolerance in the primary root growth.PA binds to targeted proteins and regulates its cellular localization,activity or downstream gene expression to participate in the response of plants to a variety of stresses,such as high salt,high temperature,freezing,and osmotic stress.In order to determine whether PA binds to PINOID(PID)protein kinase,we cloned the PID CDS region and three truncated proteins from Arabidopsis thaliana,and constructed them into the expression vector pET-28a(+)with His tag.Then we induced the expression of recombinant proteins and purified them in Escherichia coli.Results of fat-blot technology showed that PA specifically bound to PID protein,and the binding sites were localized in the truncated(1-228)amino acid residues.Moreover,results of site directed mutagenesis showed that the 119-121 three lysines(KKK)in PID protein was the key sites for PID-PA interaction.PID regulates the activity and polar localization of PIN proteins by phosphorylating plasma membrane PIN protein.We compared the effects of salt stress on the abundance of PID and PIN2 at the plasma membrane.125 mM NaCl treatment significantly reduced the abundance of PID-GFP(ProPID:PID-GFP)and PIN2-GFP(ProPIN2:PIN2-GFP)at the plasma membrane;Greater reduction of PID-GFP and PIN2-GFP was found in the pld?1 pld?-1 when exposed to salt stress.Exogenous PA(25 ?M)significantly alleviated salt inhibition.Meanwhile,we mutated KKK(binding sites of PID and PA)to GGG,and obtained WT complemention(ProPID:PID-GFP;pid-1)and mutant complemention(ProPID:mPID-GFP,pid-1)materials and treated them with salt treatment.After 125 mM NaCl treatment,compared with WT complemention,more reduction of the PID fluorescence were found in the mutant complemention at the plasma membrane,and exogenous PA restored the abundance of PID-GFP in WT complemention,but not in mutant complemention lines.These results suggest that NaCl reduces the abundance of PID and PIN2 at the plasma membrane,and PLD/PA stabilized their abundance,which may depend on the PA-PID interaction.We used 125 mM NaCl to treat WT,PID mutants(pid-1 and pid-2)and pid-1 complemention materials for 6 days,the differences of root development among different genetic materials were analyzed.The results showed that compared with WT,pid mutants showed a hypersensitive phenotype in root elongation.The primary root growth of WT complemention ProPID:PID-GFP;pid-1,but not the mutant complemention,restored to the level of WT.Exogenous PA effectively alleviated the inhibition of salt stress on the primary root length of WT and ProPID:PID-GFP;pid-1,and had no effect in pid mutant and ProPID:mPID-GFP;pid-1.Moreover,pid-1 mutant developed pin-like inflorescence,aberrant flowers,and abnormal cotyledons,with three cotyledons being the most common phenotypes;Both the WT and mutant transgene were able to complement the PID-dependent developmental defects of pid-1 mutant.Compared with WT,the pid-1 mutant exhibited reduced root gravitropism,and both ProPID:PID-GFP;pid-1 and ProPID:mPID-GFP,pid-1 recovered this phenotype.These experimental results indicate that PA-PID interaction specifically regulates PID functions in response to high salt stress,and may not be involved in regulating other developmental processes.Fat-blot experimental technology showed that PA bound to the cytoplasmic part of PIN2 protein PIN2CL,and the 496 amino acid(R)and 497 amino acid(K)were the main binding sites for PA-PIN2CL interaction.We constructed WT complemention material of the pin2-1(PropiN2:PIN2;pin2-1)and non-PA-binding complemention material of the pin2-1(PrOPIN2:mPIN2;pin2-1)and treated them with salt stress.The results showed tha,after 125 mM NaCl treatment,compared with WT,both pin2-1 and pin2-2 mutant showed a salt hypersensitivity phenotype in the growth of the primary root,and exogenous PA could not restore the phenotype of the pin2 mutant.Further studies found that the phenotypes of ProPIN2:PIN2;pin2-1 and ProPIN2:mPIN2;pin2-1 were consistent with WT under NaCl treatment.The above results show that PIN2 functions downstream of PLD/PA signal during salt stress,and the interaction of PA-PIN2 may not be directly involved in the regulation of plant salt tolerance.We used heterologous expression in Xenopus to study the regulatory effect of PA on PID-mediated PIN2 activity.The cells that expressed PIN2 alone did not detect auxin transport activity,while the co-expression of PID and PIN2 induced auxin efflux,indicating that the PIN2 transport activity was dependent on PID phosphorylation.Exogenous PA significantly increased the transport activity of PIN2,but when only PIN2 protein was expressed,PA had no effect on auxin transport.At the same time,PC did not regulate the auxin transport activity of PIN2.In addition,when co-expressed PIN2 and the mutant PID treated with PA,no PA-promoted auxin efflux tansport was found.The auxin marker DII-VENUS was used to observe the real time changes of auxin in root tips of different genetic materials.When DII-VENUS fluorescence increased,auxin content decreased.Under control conditions,compared with WT,higher level of auxin content in pldal pld?-1,pid-1,and pin2-1 were found;After 125 mM NaCl treatment,DII-VENUS fluorescence in WT root tip meristem was higher than the control,suggesting the auxin content decreased,while the auxin levels in the pldal pld?-1,pid-1,and pin2-1 were basically unchanged.Exogenous PA resulted in lower levels of root tip DII-VENUS fluorescence in the WT and plda1 pld?-1,which did not affect the auxin levels in the pid-1 and pin2-1.In addition,we also observed the DII-VENUS fluorescence in the background of ProPID:PID-GFP;pid-1 and ProPID:mPID-GFP;pid-1.The results showed that under the control conditions,there was no significant difference between the two complemention lines and WT.After NaCl treatment,the changes of the fluorescence in ProPID:PID-GFP;pid-1 and WT was similar,while similar changes of the fluorescence were found in ProPID:mPID-GFP;pid-1 and pid-1 mutant.In conclusion,under salt stress,PLDal and PLD? are activated,producing signal molecule PA,which binds to and activates the activity of PID phosphorylation of PIN2,resulting in the enhancement of the PIN2 activity in the plasma membrane,which directs the dynamic redistribution of auxin in response to salt stress.This study reveals a molecular mechanism based on phospholipid signal in regulating auxin signal transduction in plant response to high salt stress,which provides an important theoretical basis and genetic resources for the genetic improvement of salt tolerant in crops.