Study On The Role Of JEM In Regulating Jasmonate Signaling-Mediated Anthocyanin Biosynthesis

Anthocyanin is a type of water-soluble flavonoid pigments in higher plants.Because of its strong antioxidant capacity,anthocyanin plays an important role in anti-oxidative stress process in plants.Anthocyanin biosynthesis is regulated by MYB-bHLH-WD40(MBW)complex,and the activity of MBW is regulated by several factors,including light,hormone,etc.Jasmonate(JA)is one of the most important plant hormones.It has been shown that JA can significantly induce the accumulation of anthocyanin in plants,acting as a positive regulator for anthocyanin biosynthesis.Previous research also indicated that Jasmonate-Zim-domain(JAZ)proteins could interact with transcription factors in the MBW complex(such as MYB75,TT8 and GL1)to inhibit their interactions.However,how do JAZ proteins inhibit the activity of transcription factors is still largely unknown.In this study,a gene that negatively regulates JA signaling-mediated anthocyanin biosynthesis was identified,and was named as JEM(JA signaling Enhancer Mutant).Compare to the wild type,T-DNA insertion mutants of JEM(lines jem-1 and jem-2),produced higher anthocyanin under salt or drought stresses.Upon MeJA treatment,the content of anthocyanins in jem mutants was significantly higher than that of the wild type.Similar phenotype was exhibited in knockout mutants generated by CRISPR/Cas9(lines jem-cas9#1 and jem-cas9#8).Furthermore,both qPCR and RNA-seq analysis revealed higher expression of early response genes(such as MYB75,TT8,UF3GT,DFR,and ANS)in JA signaling that related to anthocyanin biosynthesis in jem-2 mutant than that in the wild type when treated with JA.These results indicated that JEM was a negative regulator of JA induced anthocyanin biosynthesis.Then JA synthesis muant opr3 and receptor mutant coil-1 were crossed to jem-2 respectively to create double mutants jem-2 opr3(Col-0)and jem-2 coil-1 for genetic analysis.Interestingly,the low anthocyanin phenotype of opr3 and coil-1 mutants were partially restored regardless of the presence or the absence of JA.These results suggested that JEM was a negative regulator in JA signaling-mediated anthocyanin biosynthesis,and acted downstream of COI1.Transient expression of GFP-fused JEM revealed a nuclear localization pattern in cells.Using truncated mutant protein,it was found that JEM-D1(1-130 aa)and JEM-D2(131-610 aa)sections were the key regions in JEM for its nuclear location.Analysis of GUS activity in JEMpro::GUS transgenic seedlings demonstrated that JEM was expressed in various tissues and organs,especially in leaves,hypocotyls and floral organs.To further investigate the regulatory mechanism of JEM,yeast two hybrid analysis was performed,and two interactive proteins of JEM were indentified,JAZ6 and JAZ8,which were further confirmed by in vitro pull-down,bimolecular fluorescence complementation(BiFC),and luciferase complementation imaging(LCI)assay.The JEM protein truncation experiments demonstrated that the D2 and D3(611?784 aa)segments were the key sections for the interactions between JEM and JAZ6,while JAZ8 and JEM interact mainly through the D3 region of JEM.Loss of function mutants of JAZ6 and JAZ8 were generated by CRISPR/Cas9(jaz6-cas9#18,jaz6-cas9#31,jaz8-cas9#4,and jaz8-cas9#10),and the anthocyanin contents in these mutants were also higher than that of wild type upon MeJA treatment,indicating that JAZ6 and JAZ8 were negative factors in JA signaling pathway that regulates anthocyanin biosynthesis.Additonaly,several WD40 domain-containing proteins were indentfied as putative JEM interactive proteins by Co-IP and tandem mass spectrometry.BiFC and point mutation analysis demonstrated that the EAR domain of JEM was the key region for the interaction.Because WD40 proteins are usually considered as transcriptional co-repressors,JEM was therefore proposed to play a role in recruiting transcriptional inhibitor proteins to form a transcriptional repression complex.In a summary,this study identifies a novel adaptor protein JEM in the JA signaling pathway that regulates anthocyanin biosynthesis,and reveals a mechanism mediated by JEM:JEM interactes with JAZ6 and JAZ8,and recruits transcriptional co-repressor protein via its EAR domain to form a transcriptional repression complex to repress the expression of genes involved in anthocyanin biosynthesis.There are also evidences showing that JEM is involved in the other physiological processes such as male gametophyte development and flowering time,indicating that JEM is likely involved in multiple transcriptional inhibitory complexes,and JEM-mediated transcriptional repression is a genral foundamental mechanism involved in plant development.The study will help us to further understand the regulatary mechanism of JA-induced anthocyanin biosynthesis,and also provide new insights in the molecular mechanism of transcriptional repression.