Integration Of RACK1 And Ethylene Signaling Regulator EIN2 Regulates Plant Growth And Development In Arabidopsis

RACK1(Receptor for Activated C Kinase1)in Arabidopsis(Arabidopsis thaliana)is a multifunctional scaffold protein encoded by three homologous genes,i.e.,RACK1 A,RACK1B and RACK1 C.RACK1 plays an important role in regulating plant growth and development,including seed germination,root development,production of rosette leaves and floral organ development.Loss-of-function of one or two RACK1 genes will causes defects in plant growth and development,and loss-of-function of all the three RACK1 genes will result in plant death.As a scaffold protein,RACK1 interacts with a variety of proteins,including kinases,phosphatases,ion channel proteins,membrane receptors,G protein and IP3(Inositol triphosphate)receptor,to regulate different activities in plants,such as plant response to abscisic acid,auxin,gibberellin and brassinolide.However,it is unclear if RACK1 is involved in the regulation of plant response to ethylene.Signal transduction of ethylene,one of the important endogenous plant hormones,is started from the perception of ethylene molecules by the ethylene receptor located on the endoplasmic reticulum membrane.Binding of ethylene by ethylene receptor leads to deactivation of the downstream CTR(Constitutive Triple Response 1)proteins,which are no long able to phosphorylate EIN2(Ethylene Insensitive 2)proteins.EIN2 proteins are then being cleaved into C-terminal and N-terminal regions,and the Cterminal regions are transported into the nucleus to activate downstream transcription factors,therefore triggers a series of physiological responses in plants,such as the ethylene triple response,and affects different aspects of plant growth and development including fruit ripening,leaf senescence,adventitious root formation,root hair development,and seed dormancy breaking.In our study,we examined the coordination of RACK1 and ethylene signalling in regulating plant growth and development.We first noticed that the phenotype of dark grown rack1 a mutant seedlings is similar to that of the wild-type Arabidopsis seedlings in response to ethylene,indicating that RACK1 may play a role in regulating ethylene signaling.We therefore examined ethylene response of the double and triple mutants of the RACK1 genes,we found that RACK1 reduntantly regulate hypocotyl enlongation and ethylene response in Arabidopsis,rack1 has shorter hypocotyl,after ethylene treatment,we found that the rack1 mutant seedlings were less sensitive to ethylene.We then generated double and high order mutants between ein2,a loss-of-function mutant of EIN2,and rack1 mutants.By examining the ein2 rack1 mutants,we found that lossof-function of EIN2 could partially restored some of the morphological phenotypes observed in the rack1 single and double mutants,such as decreased fertility,shorter root length,and smaller rosettes,but that in the rack1 a rack1b rack1 c triple mutant remained largely unaffected by loss-of-function of EIN2.On the other hand,compared with ein2 and corresponding rack1 mutants,ein2 rack1 mutants produced more rosette leaves and showed a delayed flowering phenotype.We also found that the phenotypes of curled leaves and twisted petioles observed in the ein2 mutants were enhanced in ein2 rack1 mutants.However,our experimental results of yeast two-hybrid indicated that EIN2 is not able to physically interact with RACK1 directly.On the other hand,RT-PCR(Reverse Transcription-Polymerase Chain Reaction)and q RT-PCR(Quantitative RT-PCR)results showed that the expression level of EIN2 was reduced in the rack1 mutants.Nevertheless,our results indicated that RACK1 coordinated with EIN2 to regulate plant growth and development in Arabidopsis.