Functional Analysis Of Zinc Finger Protein ZFP181 From Rice(Oryza Sativa L.)and Glutamate Receptor-like Proteins GLR3.3 And GLR3.6 From Arabidopsis

Drought stress is a major limiting factor for crop production.Under drought stress,plants alter their physiology,such as root structure,stomatal opening and closing status,often accompanied by early flowering,stunted growth,and then serious reducing crop yields.According to the report of the FAO,crop losses due to drought account for 30%.Over these years,with the efforts of breeding workers,the yield of cultivated rice in China has increased significantly.Therefore,to produce high-yield,drought-resistant crops will be a new challenge for us.Further studies of molecular mechanisms combined with the development of biotechnologies will facilitate breeding drought-tolerant crop varieties.Plant A20/AN1 zinc finger protein plays an important role in abiotic stress response.The ubiquitin ligase activity of A20/AN1 zinc finger proteins in animals is closely related to their biological functions.More and more plant A20/AN1 zinc finger proteins are reported to have ubiquitin ligase activity,and protein ubiquitination modification is a universal posttranslational modification that plays an important role in all processes of plant growth and development.Therefore,it will be a good entry point to reveal how they participate in the stress response from the changes of ubiquitination level.Preliminary work in our laboratory has shown that rice A20/AN1 type zinc finger protein gene ZFP181 overexpression transgenic plants are more tolerant to drought stress,and ZFP181 can interact with OsLEA 19,one of the late embryogenesis abundant proteins.In this study,we analyzed the molecular mechanism of ZFP181 in regulating rice drought stress response.The main findings are as follows:1.In vitro ubiquitination experiment proved that ZFP181 has ubiquitination ligase activity.Analyzing the data from Genevestigator website,it was found that rice LEA family genes,including OsLEA 19,are generally induced by drought stress,and using OsLEA19 specific antibodies revealed that OsLEA 19 protein level is very low in wide type plants under normal conditions,but with 20%PEG or dehydration stress treatment,the protein level will increase significantly,indicating that both transcription and translation levels of OsLEA19 could be significantly induced under drought stress.Combined with the reported results that OsLEA19 overexpression transgenic plants are more tolerant to drought stress,indicating that OsLEA19 is involved in regulating drought stress response.Furthermore,it was found that ZFP181 overexpression transgenic plants accumulated OsLEA19 significantly in vivo.Cellfree experiment showed that OsLEA19 protein was mainly degraded by the proteasome pathway,but ZFP181 could not influence the degradation rate of OsLEA19 in this experiment,and in vitro experiments,we failed to detect the ubiquitination modification of OsLEA19 by ZFP181.It is speculated that ZFP181 positively regulates the rice drought response by accumulated OsLEA19 in an unknown way.2.K-ε-GG specific antibody was used to detect the ubiquitination level of protein in wide type with the normal condition and drought treatment,and in ZFP 181 transgenic plants under normal condition.Ubiquitination affects many biological processes including transcription and translation.Drought treatment mainly leads to an increase in the ubiquitination level of pathway proteins including glycolysis and metabolism,and a decrease in the ubiquitination level of pathway proteins such as signal transduction and posttranslational modification.The ubiquitination level of the protein in ZFP181 transgenic plants is mainly reduced,which include signal transduction,post-translational modification and other pathway proteins.The proteins co-regulated by ZFP 181 and drought are mainly involved in ubiquitination-related and endocytosis pathways.It is speculated that ZFP181,as an E3 ubiquitin ligase,participates in drought stress response by changing the ubiquitination levels of ubiquitination-related proteins,endocytosis-related proteins,and BAG family proteins.The occurrence of environmental stress such as drought is often accompanied by the occurrence of insect pests.FAO found that disasters such as locusts can affect 20%of the total cultivated land area in the world,such disasters are also serious in China.Indicating that insect pests are another severe limiting factor that affecting plant growth and yield.Plants mainly defend against herbivore and nematode attacks by sending signals from the wounded sites to the whole plant.But the mechanism of how plants produce and transmit these rapidly moving long distance signals(referred to as systemic wounding signals)is still not clear yet.Further studies of its molecular mechanisms are important for plant surviving from these disasters.Based on previous work,this study analyzed the important role of glutamate receptor-like proteins GLR3.3 and GLR3.6 in the root-to-shoot long-distance signal transmission.The main results are as follows:1.Using transgenic plants which expressing the sensitive calcium sensor GCaMP6s,it was found that cutting the main root of Arabidopsis thaliana triggers the calcium waves from the root to all shoot part leaves,and application of glutamate at the wounding site triggers a similar response,but these phenomena disappeared in glr3.3glr3.6 mutant.Using the surface potential detection method,it was found that cutting the main root triggers the electrical signal from the root to all shoot part leaves,and the application of glutamate at the wounding site triggers a similar reaction,but these phenomena cannot be found in glr3.3glr3.6 mutant.Through grafting experiments,it was found that when the wild type was used as rootstock and glr3.3glr3.6 was used as scion,wounding and glutamate could not trigger root-to-shoot calcium and electrical signals.When glr3.3glr3.6 was used as rootstock,wide type was used as scion,wounding and glutamate can still trigger root-to-shoot calcium and electrical signals.These results indicate that GLR3.3 and GLR3.6 are mainly involved in the transmission of these wounding-and glutamate-induced root-to-shoot calcium and electrical signals.It was further found that in the AHA1 constitutive expression mutant ost2-2D,the depolarization duration of wounding-and glutamate-induced root-to-shoot electrical signals are significantly decreased,indicating that AHA1 is also involved in wounding-and glutamateinduced root-to-shoot electrical signals.2.When using single-cell calcium imaging experiments in HEK293T cells coexpressing GCaMP6s and plant GLR to investigate the calcium channel activities of GLR3.3 and GLR3.6,it was found that 100 mM glutamate with a pH lower than 7.5 cannot activate GLR3.3 and GLR3.6,and 100 mM glutamate with a pH higher than 7.5 can activate GLR3.3 and GLR3.6,causing a large calcium influx.It was further found that when the pH is higher than 7.5 but the glutamate concentration is decreased to 25 mM or 10 mM,GLR3.3 and GLR3.6 cannot be activated.These two channels can only be activated when the pH is higher than 7.5 and the glutamate concentration is higher than 50 mM.The single-cell patch-clamp experiment further proved that the inward current of cations can only be recorded in HEK293T cells expressing GLR3.3 or GLR3.6 when the pH is higher than 7.5 and the glutamate concentration is higher than 50 mM.This indicates that Arabidopsis GLR3.3 and GLR3.6 are high pH-dependent and high glutamate concentration-dependent.Based on these results,it is speculated that when the plant is wounded,the proton pump is inhibited first,initiating apoplastic alkalization that,together with wound-released glutamate,activates GLR3.3 and GLR3.6,causing a large calcium influx,resulting in depolarization of membranes and generates an electrical signal,and then the P-type proton pump(AHA1)is reactivated,which further closes the GLR channels and repolarizes the membrane,thereby conducting a systemic wounding signal.