The Endocytosis Of AtRGS1 Is Involved In Regulation Of JA And PAMPs/DAMPs Signaling In Arabidopsis

Signal transduction through a heterotrimeric G protein complex,classically consisting of G?,G?,and G? subunits,is an essential plasma membrane(PM)signaling pathway in most eukaryotes.In Arabidopsis,heterotrimeric G proteins regulate diverse plant growth and defense processes by coupling to 7TM AtRGS1(Regulator of G-protein signaling protein 1).In the present study,various cellular techniques,plant physiological and molecular methods were used to reveal the endocytosis and PM dynamics of AtRGS1 upon jasmonate(JA)or PAMPs/DAMPs treatment.(1)G?-deficient mutant(gpa1-4),G?-deficient mutant(agb1-2),and double mutant gpa1-4/agb1-2 displayed reduced sensitivity to JA in root growth,anthocyanin accumulation,and LOX2 gene expression.These findings suggest that both G? and G?subunits likely function in some,but not all,JA responses as positive regulators.(2)LSCM and VA-TIRFM analysis showed that JA induced the endocytosis of AtRGS1 in a time-dependent manner and shorterned the residence time of AtRGS1 on the PM.Furthermore,by using the protein kinases inhibitor K252 a and a C-terminal truncation mutant AtRGS1-?Ct-YFP,we demonstrated that JA induced the endocytosis of AtRGS1 in a phosphorylation-and C-terminus-dependent manner.In additioon,Western blot analysis displayed that the internalized AtRGS1 proteins did not degrade,which might recycle to the PM.(3)FRET-FLIM assay was performed on the transient co-expressed Nicotiana benthamiana leaves with AtRGS1-GFP and AtGAP1-mCherry to analyze their interaction.By determining the donor fluorescence lifetime and the FRET frequency,we found that AtRGS1 directly interacted with AtGPA1 under steady-state condition and that JA treatment triggered the dissociation of AtRGS1 and G protein pre-formed complex.(4)Using VA-TIRFM and SPT analysis,the lateral mobility of AtRGS1 within the PM upon JA treatment was quantitated.AtRGS1 proteins exhibited comparable trends to increased lateral mobility.The phosphorylation state and C-terminus of AtRGS1 proteins affected their lateral mobility regulated by JA on the PM.(5)LSCM and VA-TIRFM analysis presented that three PAMPs/DAMPs(flg22,chitin and PIP1)induced the endocytosis of AtRGS1 in a time-dependent manner and shorterned the residence time of AtRGS1 on the PM.Furthermore,by using the protein kinases inhibitor K252 a and a C-terminal truncation mutant AtRGS1-?Ct-YFP,we demonstrated that three PAMPs/DAMPs induced the endocytosis of AtRGS1 in a phosphorylation-and C-terminus-dependent manner.(6)FRET-FLIM assay was performed on the transient co-expressed Nicotiana benthamiana leaves with AtRGS1-GFP and AtGAP1-mCherry to analyze their interaction.By determining the donor fluorescence lifetime and the FRET frequency,we found that AtRGS1 directly interacted with AtGPA1 under steady-state conditions and that three PAMPs/DAMPs treatment triggered the dissociation of AtRGS1 and G protein pre-formed complex.(7)Using VA-TIRFM and SPT analysis,the lateral mobility of AtRGS1 and AtRGS1-?Ct within the PM upon three PAMPs/DAMPs treatment was quantitated.Full-length AtRGS1 and C-terminus-truncated AtRGS1 proteins had similar variations in motion range and showed the opposite behavior for diffusion coefficients.Taken together,these findings suggest that JA and three PAMPs/DAMPs signals activate heterotrimeric G proteins through the endocytosis of AtRGS1 and dissociation of AtRGS1 from AtGPA1,thus providing valuable insight into the mechanisms how the G protein system perceives and transduces extracellular signal to regulate JA and PTI responses.