Arabidopsis Phosphoinositide-specific Phospholipase C4 Negatively Regulates Seedlings Salt Tolerance

Phosphoinositide-specific phospholipase C(PI-PLC)is a class of enzyme that hydrolyzes phosphotidylinositol 4,5-bisphosphate(PIP2)into two second messengers inositol 1,4,5-tris phosphate(IP3)and diacylglycerol(DAG).In plant,IP3 or its derivatives induces the release of Ca2+ from intracellular stores.Ca2+ acts as signal molecular and participates in numerous developmental and adaption processes in plants.DAG is rapidly phosphorylated to PA by diacylglycerol kinase(DGK)and the PA has typically emerged as the second messenger.PIP2,IP3,DAG and PA involved in various stesses response.Therefore,the study of the plant PI-PLC is important to reveal the mechanism of the plant growth and development and the resistance to various stresses.Salt stress is one of the major abiotic stresses and severely reduces plant growth and crop productivity worldwide,and it is crucial to unravel the plant salt tolerance mechanism.Salt stress stimulates the rapid accumulation of PIP2,IP3,DAG and PA in plant cells.Previous physiological and pharmacological studies have suggested that the activity of PI-PLC plays important roles in regulating plant salt stress responses by altering intracellular Ca2+ concentration.These results implied that PI-PLC might involve in regulating plant salt tolerance.However,the individual member of plant PI-PLCs that involves in the process is still needed to be identified.Salt stress showed the mostly effective induction on AtPLC4 transcription in the nine PI-PLC genes in Arabidopsis seedlings.AtPLC4 was the second highest expressed PI-PLC.These results implied that AtPLC4 might involve in regulating plant salt tolerance.We found that AtPLC4 was expressed constitutively along the growth stages in all the tested tissues.We also found that AtPLC4 was primarily localized at the PM and NaCl treatment resulted in an increase in AtPLC4 at the PM of root cells and the nuclear of root tip cells.Since the AtPLC8 and AtPLC9 were the two genes which share the highest homology with AtPLC4 among the nine PI-PLCs and their transcriptions were not induced by salt stress.The growth phenotypes of plc4,plc8,plc9 and plc4 complementation seedlings grown under salt stress condition were analyzed.The results showed that loss of AtPLC4 but not AtPLC8 and AtPLC9 function might lead seedlings hyposensitive to salt stress.AtPLC4-overexpression seedlings were hypersensitive to salt stress.In addition,we analyzed the sensitivity of plc4 and AtPLC4-overexpression seedlings to NaNO3,KCl and mannitol.The results suggested that both the hyposensitivity of plc4 seedlings and the hypersensitive of AtPLC4-overexpression seedlings to salt stress were the specific response to Na+ but not Cl-or osmotic stress.AtPLC4 inactive form overexpression seedlings did not exhibit any visible growth phenotypic difference comparing with Vector seedlings under salt stress.This result suggested that the PI-PLC activity was required for the AtPLC4-overexpression-induced salt stress hypersensitivity.Aequorin luminescence-based cytosolic Ca2+ imaging analyses revealed the AtPLC4 overexpression seedlings with aequorin expressing showed an enhanced steady-state cytosolic Ca2+ and the salt-stimulated increase in cytosolic Ca2+ after AtPLC4 induction,and the enhanced salt-stimulated increase in cytosolic Ca2+ in AtPLC4 overexpression seedlings was eliminated by EGTA.Additionally,the hypersensitivity of AtPLC4-overexpression seedlings to salt stress was partly rescued by EGTA.We found that the transcription levels of stress-responsive genes,RD29B,ZAT10 and MYB15,were regulated by AtPLC4.To further dissect the physiological function of AtPLC4 in response to salt stress,we analyzed Na+ and K+ accumulation in plc4 and AtPLC4-overexpression seedlings under salt stress.The results showed that plc4 mutant seedlings accumulated more Na+ and K+,and AtPLC4-overexpression seedlings accumulated less Na+ and K+.Our findings suggest that AtPLC4,a Phosphoinositide-specific phospholipase C,negatively regulates Arabidopsis seedlings salt tolerance,and RD29B,MYB15 and ZAT10 genes and Ca2+ may involve in this regulating process.AtPLC4 also regulates the Na+ and K+ accumulation in Arabidopsis seedlings during salt stress.These findings not only provide a new experimental evidence for plant salt tolerance mechanism,but also expand the function of plant PI-PLC in Arabidopsis.