Transporter-mediated Nuclear Entry Of Jasmonoyl-isoleucine Is Essential For Jasmonate Signaling

In both animals and plants,small molecule hormones play pivotal roles in coordinating organismal growth,development and immunity by transcriptional reprogramming,which requires the binding of the hormonal ligands with their cognate receptors either extranuclearly and nuclearly.The entry of receptor-hormonal ligand complex through the nucleocytoplasmic transport machinery is well established,whereas the mechanism regulating nuclear entry of the hormone molecules remains enigmatic.Here,we identified and characterized a putative Arabidopsis jasmonate(JA)transporter AtJAT1.The main results are as follows:To identify the putative JA transporter AtJAT1,we established a heterologous system in yeast cells based on our finding that exogenous JA inhibited yeast cell growth.The AtJATl-GFP signal was primarily localized in the plasma membrane(PM)of yeast cells.3H-JA export assays in yeast cells show that AtJATl functions as jasmonate transporter.In addition,their transport activities were inhibited by the inhibitors of ABC transporters.We characterized an Arabidopsis mutant,atjatl,in which AtJAT1 expression was fully disrupted by a T-DNA insertion.The atjat1 plants exhibited a larger stature at both vegetative and reproductive stages including larger flowers and seeds.The large stature of atjat1 plants is caused by the enlarged size of cells but not the increased number of cells.After treated by exogenous JA,Jasmonoyl-Isoleucine(JA-Ile)and Coronatine(Cor),the growth inhibition of primary roots in wild-type(accession Columbia,Col)plants was relieved in the atjat1 mutant,confirming that JA signaling is inhibited by disrupting AtJAT1 expression.The transcript level of the JA-responsive genes(OPR3,JA25,JA27,MYB21 and MYB24)was significantly lower in the atjatl than in Col plants.GUS analysis revealed weak activities of the AtJAT1 promoter in vascular tissues of cotyledons and in floral organs including stamens and flower receptacles.The promoter activities were greatly enhanced by exogenous MeJA treatment.Consistently,the AtJAT1 transcript abundance increased upon MeJA treatment.Compared to Col,the growth of Botrytus cinerea and P.syringae was greatly enhanced in atjat1 plants.Collectively,these results show that AtJAT1 is required for activating the COI1-depedent core JA signaling pathway.We further detected the AtJAT1-GFP signal in the plasma membrane(PM)and nuclear envelope(NE)of Arabidopsis root cells.3H-JA-Ile uptake assays in the nuclei of Col,the atjat1 mutants,transgenic plants over-expressing AtJAT1(JAT1OX)showed that AtJAT1 functions as a high-affinity transporter for the nuclear import of JA-Ile.The Km value for AtJAT1-mediated nuclear uptake JA-Ile is 166.4 nM.In additional AtJAT1-mediated nuclear import of 3H-JA-Ile was inhibited by the inhibitors of ABC transporters and was also dependent on ATP.Substrate competition showed that JA-Ile and Cor are the transport substrates of AtJAT1 in the NE.Electron microscopic autoradiography was performed to investigate the partition of JAs(Jasmonates)in callus cells.Compared to Col cells,jar1 cells exhibited a greatly decreased abundance of silver particles in the nucleus but a comparable silver particle abundance in the cytoplasm,showing that the silver particles in the nucleus represents predominantly 3H-JA-Ile.As expected,compared to Col cells,the atjatl cells showed greatly lower silver particle abundance in the nucleus,confirming an in vivo role of AtJAT1 in mediating nuclear entry of JA-Ile.In contrast to the decreased 3H-JA-Ile in the nucleus,the atJat1 cells exhibited significantly higher silver particle abundance in the cytoplasm than that in Col cells,indicating that AtJAT1 also mediates cellular export of JA/JA-Ile across the PM.To confirm an in planta role of AtJATl in mediating nuclear entry of JA-Ile,we next examined the degradation of JAZ proteins.After treating with exogenous JA,the JAZ1-GFP signal was persistent in the atjat1 root cells,confirming an inhibition of JA signaling in the atjat1 cells.The JAR1/jar1;AtJAT1/jat1 hemizygous F1 plants derived from a cross between the jar1 and the atjat1 mutants exhibited the characteristic defects in late stamen development and were sterile.These results supports that both cytoplasmic synthesis and nuclear import of JA-Ile are essential to maintain a critical nuclear JA-Ile concentration for activating JA signaling.3H-JA export assays in the suspension cells of Col,the atjat1 mutants,transgenic plants over expressing AtJAT1(JAT1OX)showed that AtJAT1 functions as a high-affinity transporter for the cellular export of JA.The Km value for AtJAT1-mediated cellular export JA(348.2 nM)is-2 fold higher than that of nuclear import of JA-Ile(166.4 nM).AtJAT1-mediated cellular export of 3H-JA is inhibited by the inhibitors of ABC transporters.Substrate competition showed that JA is the preferred transport substrate of AtJAT1 in the PM.In summary,these results demonstrate that AtJATl mediats,beside nuclear entry of 3H-JA-Ile,cellular export of 3H-JA across the PM in Arabidopsis cells,which is essential for regulating the highly dynamic JA signaling.