A Peroxisomal Jasmonate Exporter Regulates The Homeostasis Between Jasmonoyl-isoleucine And Jasmone

Peroxisomes are the hub of cellular metabolism and involved in the biosynthesis of a number of phytohormones including auxin,jasmonic acid(JA),benzoic acid(BA).Albeit being long proposed,peroxisomal transporters mediating the efflux of these phytohormones and their essential roles in regulating phytohormone signaling remain to be illustrated.JA are a family of phytohormones that regulate plant growth,development,and responses to stresse.JA is derived from the oxygenation of?-linolenic acid released from the plastid membrane.The plastid-generated JA precursor,12-oxophytodienoic acid(OPDA)is translocated to the peroxisomes by COMATOSE(CTS)where JA is synthesized after reduction by OPDA reducdase 3(OPR3)and three round ?-oxidation.However,mechanisms on the peroxisomal efflux of JA is unclear.Arabidopsis thaliana JA transporters 2(AtJAT2)is involved in JA import in yeast cells and mediates the export of peroxisoal JA in Arabidopsis.To identify the putative AtJATs,we established a heterologous system in yeast cells(Saccharomyces cerevisiae strain YPH499)based on our finding that exogenous JA inhibited yeast cells growth.AtJAT2 transformant cells exhibited an enhanced sensitivity to exogenous JA and led to up to 2.5-fold increase of 3H-JA retention in the uptake assays,indicating that AtJAT2 is able to facilitate JA import across the plasma membrane in the yeast cells.As promoter activities of OPR3,the promoter activities of AtJAT2 were also restricted to the vascular tissues of seedlings including roots,leaves,and of the floral organs including stamens,sepals and petals.AtJAT2-GFP signal was colocalized with that of PTS1(peroxisomal targeting signal)-RFP,a marker for peroxisome targeting,indicating that AtJAT2 is localized in the peroxisomes.Furthermore,immunoblotting showed that AtJAT2-GFP protein is detected in the peroxisomal membrane.JA transport assays in the isolated peroxisomes of Arabidopsis cells by3 H-JA showed that AtJAT2 is a high-affinity(Km=137.5±38.6 nM)transporter mediating the export of JA across the peroxisomal membrane.This AtJAT2-mediated peroxisomal export of 3H-JA is inhibited by the inhibitors of ATP binding cassette(ABC)transporters and is dependent on ATP.AtJAT2 is involved in the regulation of core JA signaling.Similar to ctsl mutants that exhibited generally normal fertility,atjat2 mutants exhibited no phenotypical alternation and normal late stamen development.The atjat2-1;cts1 double mutants,however,exhibited the characteristic defects in late stamen development and were sterile.Exogenous MeJA application could rescue the defects of the double mutants in late stamen development and also restored the fertility,indicating that AtJAT2 and CTS function synergistically to regulate JA signaling.The peroxisomes of atjat2 cells accumulated higher level of JA than those of Col cells.In contrast,the JA and JA-Ile level were reduced in the leaf cells of atjat2 mutants.The expression of the early JA-responsive genes decreased significantly in both atjat2-1 and atjat2-2 plants.JAZ1-GFP signal in the nuclei of root cells were stronger in the atjat2-1 than in Col background.However,exogenous JA triggered the disappearance of the JAZ1-GFP signal in the nuclei of both Col/JAZ1-GFP and atjat2-1/JAZ1-GFP plants.Relative to Col plants,the growth of B.cinerea and A.brassicicola is greatly enhanced in atjat2 mutant.Taken together,these results indicate that AtJAT2 involved in regulating JA-Ile production by modulating the cytosolic availability of JA.AtJAT2 is involved in regulating the metabolic flux between JA-Ile and CJ by modulating the distribution of JA between the cytosol and peroxisomes.The atat2 cells showed a higher cis-jasmone(CJ)content than Col cells,whereas CJ level in leaf cells of opr3 plants,was greatly reduced.In consistence,an elevated transcript level of CJ-inducible OPR1 and KMD3 genes was also observed in atjat2 mutants.We tested the repellence of aphids by olfactometer bioassay,the atjat2 plants attracted less aphids than Col plants,whereas opr3 plants attract significantly more aphids.These results indicate that AtJAT2 is involved in regulating CJ production by controlling the distribution of JA in the peroxisomes.Furthermore,both the promoter activity and the transcription of AtJAT2 was induced by exogenous JA,but inhibited by exogenous CJ treatment.In summary,these results indicate that AtJAT2-mediated JA distribution between the peroxisome and cytosol play important roles in regulating not only JA signaling but also the metabolic flux distribution between JA-Ile and CJ.Thus,a novel role of AtJAT2 in orchestrating development/immunity with plant-insect interactions is proposed.