Phosphoinositide-Specific Phospholipase C9(PI-PLC9) Is Involved In Thermotolerance And Heat Shock Signal Transduction In Arabidopsis

Temperatures above the normal optimum are sensed as heat shock(HS)by plants.Every year worldwide severe agricultural losses are attributed to heat,often in combination with drought and other stresses.Thus,how the sessile plants sense the thermal signal and how to improve the heat resistance of plants become a pressing issue for us.Heat stress response(HSR)is one of the most significant adaptation ability for plants.Many signaling pathways has been involved in heat shock response.Calcium as a principal second messenger has been demonstrated is involved in the HSR of plants.HS induces rapid increase of intracellular calcium concentration.There are two different pathway for intracellular calcium concentration changing.One is extracellular calcium through the calcium channels on the plasma membrane influx into the cytoplasm.The other way is calcium outflow from intracellular calcium stores into the cytoplasm.It has been reported that there is ion channels on plasma membrane for calcium influx into intracellular in response to HS in moss plants.Heat shock(HS)has been reported can activate phosphoinositide-specific phospholipase C(PI-PLC)in animals and plants.In this study we used the T-DNA knockout mutants to provide direct molecular genetic evidence for PI-PLC play a significant role in the early respond of HS signal pathway in Arabidopsis thaliana.T-DNA knockout mutants for Arabidopsis thaliana PI-PLC(AtPLC)were obtained and their thermotolerance were observed.There were no different phenotype for AtPLC mutants compared with wild type seedings under normal growth condition.Nevertheless,the atplc9 displayed a serious decline thermotolerance compared with wild type after HS.Expression of the full-length AtPLC9 gene under native promoter in atplc9 could rescue the heat-sensitive phenotype.The overexpression lines exhibited much higher heat resistance than wild type and vector control.GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that A tPLC9 expression was ubiquitous.The fluorescence distribution of the fusion protein AtPLC9 promoter:A tPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the periphery of root cells.Results from the PLC activity assay showed that there was a prevention of inositol 1,4,5 trisphosphate(IP3)accumulation in atplc9 during HS.The IP3 generation in the overexpressing lines was improved.The heat-induced calcium releasing from intracellular store in HS pathway was decreased in atplc9.The accumulation of heat shock protein 18.2 and 25.3(HSP18.2,HSP25.3)were downregulated in atplc9 and upregulated in AtPLC9 overexpression lines after HS.Together,these results indicate that AtPLC9 play a key role in HS signal transduction and thermotolerance of Arabidopsis.Taking all of our data into account,we hereby modify our Ca2+/CaM HS signaling pathway as follows:HS activates PLC,causing an increase in IP3 and Ca2+release,which then activates and promotes the expression of AtCaM3,which in turn activates AtCBK3 kinase to phosphorylate AtHSFA1a,thereby affecting the accumulation of HSPs and thermotolerance in Arabidopsis.