Molecular Mechanism Of Jasmonic Acid Precursor Transport And Its Signal Transduction In Regulating Secondary Wall Synthesis In Populus

Fast-growing forest trees have important ecological and economic values.They produce wood which is widely used in bioenergy production,paper and furniture manufacturing.Secondary cell walls are the main components of wood,which provides mechanical support to plant growth and gives the xylem the ability to transport water and inorganic salts over a long distance.On another hand,the secondary walls also improve the ability to defense pests and disease in plants.Therefore,it is very important to study the mechanism of secondary wall synthesis and genetic regulation in increasing wood yield and improving traits.It has been shown that secondary wall synthesis was affected by seasonal factors and external stresses.The phytohormone jasmonic acid(JA)was widely involved in these stress response mechanisms,however,the molecular mechanism by which JA affects secondary wall synthesis remains unclear in woody plants.JA is an important stress hormone molecule that plays a key role in plant development and growth,stress and defense responses.Previous studies have shown that JA biosynthesis starts in chloroplasts,where the JA precursor 12-oxophytodienoic acid(OPDA)is synthesized,and then OPDA is transported across the membrane to the cytoplasm,where JA is synthesized in the peroxisome.At present,the functional studies of JA are mainly concentrated in Arabidopsis and rice,and the research in woody plants is relatively lagging behind,and the synthetic pathways and their regulatory mechanisms are still poorly understood.Poplar has become one of the most widely planted fast-growing tree species in the world because of its widely distribution,fast growth,easy cuttings and high lumbering rate.The completion of whole genome sequencing of Populus trichocarpa and the establishment of poplar genetic transformation system have laid a solid foundation for the study of secondary growth of poplar.In this study,we used bioinformatics and molecular genetics identified an OPDA transporter protein,OPDAT1,localized on the chloroplast inner membrane in poplar,and the effect of JA precursor transport on secondary wall synthesis was confirmed using OPDAT1 transgenic poplar as material.The molecular mechanism of JA signal transduction regulating secondary wall synthesis in poplar was analyzed by overexpressing the JA signal repressor gene JAZ(JASMONATE ZIM Domain).The main findings are as follows:(1)Cloning of poplar JA precursor transporter protein gene OPDAT1 and its expression analysisPrevious studies have demonstrated that the transmembrane transporter protein FAXs(fatty acid export)of Arabidopsis thaliana is involved in the transmembrane transport of fatty acids.Based on amino acid sequence homology,screening of nine members highly homologous to Arabidopsis FAX genes from the genome of Populus trichocarpa and they are named Ptr FAX1.1,Ptr FAX1.2,Ptr FAX2.1,Ptr FAX2.2,Ptr FAX3(OPDAT1),Ptr FAX4.1,Ptr FAX4.2,Ptr FAX5 and Ptr FAX6.Analysis of the promoter elements of these genes revealed the presence of seven jasmonic acid response elements(CGTCA)on the promoter sequence of OPDAT1 with concentrated element positions.Gene expression analysis confirmed that OPDAT1 expression could be rapidly induced by injury signals,suggesting that OPDAT1 may be involved in the jasmonic acid signaling pathway.GUS staining analysis showed that OPDAT1 was highly expressed mainly in leaves and stems,and the expression in leaves showed a positive correlation with chloroplast content.In addition,the OPDAT1: GFP fusion gene was transiently expressed in tobacco protoplasts,and co-localization analysis with the chloroplast endosomal protein gene At PIC1: RFP revealed that OPDAT1 was localized on the endosomal membrane of chloroplasts.The above results suggest that OPDAT1,as a transporter protein localized on the chloroplast inner membrane,may be involved in the transmembrane transport of the JA synthetic precursor OPDA outwards from in the chloroplast.(2)Heterologous yeast expression system confirms that OPDAT1 has a transport function for OPDAIn order to verify the transmembrane transport function of OPDAT1 to OPDA,yeast expression vectors of OPDAT1 were constructed and transferred into wild type and transporter deletion mutant yeast cells respectively.The results show that exogenous addition of OPDA can inhibit the growth of wild-type yeast cells,but overexpression of OPDAT1 can reduce its inhibitory effect.After external addition of OPDA,the OPDA content in yeast was measured at different time points.The results showed that after overexpression of OPDAT1,the content of OPDA in yeast cells was significantly lower than that of control yeast cells expressing the empty vector.Similarly,pretreatment of yeast cells with OPDA,the OPDA content in yeast cells overexpressing the target gene rapidly decreases and the rate of loss is significantly higher than that of wild-type yeast.All of the results indicate that OPDAT1 has a transport function for OPDA in yeast cells.(3)Poplar OPDAT1 can export OPDA from chloroplasts and affect JA biosynthesisTo determine the biological function of OPDAT1 protein in poplar and whether it affects JA synthesis,OPDAT1 overexpression and CRISPR-cas9 knockdown vectors were constructed and transformed to wild-type poplar,the transgenic plants with overexpression and loss-of-function were obtained,respectively.After analysis,it was found that in the opdat1 mutant,the OPDA content in leaf chloroplasts was significantly higher than that in wild-type plants,but the JA content in leaves was significantly lower than that in wild-type plants.Transcriptome data analysis confirmed that the expression of genes related to JA synthesis and signal transduction(LOX2,JAZ5,etc.)was significantly down-regulated in opdat1 gene mutant plants.Since injury can induce the rapid accumulation of JA in plants,changes of OPDA and JA contents were detected by mechanical damage treatment of leaves of transgenic plants.The results showed that the injury treatment resulted in significantly higher up-regulation of chloroplast OPDA content in opdat1 mutant leaves than in wild-type plants,but significantly lower JA content than in wild-type plants.RT-q PCR analysis revealed a consistent expression changes fashion of JA synthesis and signal transduction-related genes in opdat1 mutant plants showed a consistent trend.The above results suggest that OPDAT1 is involved in the transport process of poplar OPDA across the chloroplast membrane and affects JA synthesis as well as JA-mediated defense responses.(4)Effect of JA precursor transport on secondary cell wall synthesis in poplarIt has been shown that lignin content increases rapidly in plants when they are attacked by pests and diseases to improve plant defenses.Exogenous application of methyl jasmonate(Me JA)in Arabidopsis promotes secondary wall thickening and lignin deposition.To investigate the role of jasmonate precursor transport on secondary cell wall synthesis in poplar,tissue sections of stems from transgenic plants were observed using OPDAT1 transgenic poplar as material.Toluidine blue staining and scanning electron microscopy showed that the secondary cell wall thickness of phloem fibers and xylem cells in the stems of opdat1 mutant plants was significantly lower than that of wild-type plants,and exogenous Me JA treatment was able to revert this phenotype.The gene expression level showed that the expression changes of key enzyme genes for secondary wall synthesis were coincided with the phenotypic changes in the secondary wall,implying that JA precursor transport affects the synthesis of the secondary wall in poplar by altering JA content.(5)Inhibition of JA signaling affects secondary wall biosynthesis in poplarTo investigate whether JA signaling regulates secondary wall synthesis,JAZ overexpression lines were used to test their function of inhibiting JA signaling.After tissue-specific expression analysis and phylogenetic tree analysis,JAZ5 overexpression vector was selected and the construct was transformed in poplar.Tissue sections of stems showed that overexpression of JAZ5 reduced the secondary wall thickness in xylem duct cells and fiber cells.RT-q PCR results showed that JAZ5 overexpression inhibited the expression level of secondary wall synthase genes.Exogenous application of Me JA was able to revert the phenotype of secondary wall phynotype in JAZ5 overexpressing plants.The above results demonstrate that JAZ5 can regulate secondary wall thickening and lignin deposition by affecting the expression of secondary wall synthesis-related enzyme genes.(6)JAZ5 interacts with WND and MYB transcription factors to regulate secondary wall synthesis in poplarPrevious studies have demonstrated that JA functions mainly through the interaction of the signal transduction repressor JAZ protein with other transcription factors.A regulatory network of poplar secondary wall synthesis dominated by NAC and MYB transcription factors has been demonstrated.Therefore,to further investigate the molecular mechanism of JA signaling regulating secondary wall synthesis,the PGBKT7 vector for JAZ5 protein and the PGADT7 vector for secondary wall switch genes of WNDs and MYBs in poplar were constructed in this study,and through yeast two-hybrid screening,we found JAZ5 interacted with MYB2,MYB3,MYB74,WND3 A,WND4A,WND6 A and WND6 B.With gene tissue expression profiling and co-localization analysis,poplar JAZ5/MYB3 and JAZ5/WND6 A interaction combinations were selected as representatives for the further study.The existence of interactions between JAZ5 and MYB3/WND6 A in plant cells were further confirmed by fluorescent bimolecular complementation assay and immunoprecipitation assay.To clarify the structural domains of JAZ5 interacting with MYB3 and WND6 A,based on the structural features of the protein sequence of JAZ5 fusion proteins of different lengths were constructed,and it was shown Jas structural domain in the JAZ5 protein was required for its interaction with MYB3 or WND6 A using yeast two-hybrid experiments.These results imply that JAZ5 can regulate secondary wall synthesis by interacting with the secondary wall switches WND6 A and MYB3 transcription factors.(7)JAZ5 inhibit the transcriptional activation of downstream secondary wall synthase genes by interacting with MYB3 or WND6ATo clarify the molecular mechanism of JAZ5 in regulating downstream secondary wall synthesis through interacting with MYB3 and WND6 A,JAZ5 and MYB3 or WND6 A were co-expressed with the expression vector,the construct of the promoter of secondary wall synthesis key enzyme genes drive GUS reporter gene in tobacco cells,and we found that MYB3 or WND6 A alone could activate the expression of CCo AOMT1,CCR2 and COMT2,but simultaneously the expression of JAZ5 and MYB3 or WND6A inhibited the expression activation of genes critical for lignin synthesis,and exogenous application of Me JA was able to relieve this inhibition.These results suggest that JAZ5 can regulate secondary wall biosynthesis by inhibiting the function of MYB3 or WND6A.In summary,this paper identified the JA precursor transporter protein OPDAT1 in poplar,which mediates the efflux of chloroplast OPDA and is involved in JA synthesis.The JA precursor transporter was shown to regulate secondary cell wall thickening and lignin deposition in poplar by altering JA content,and the JA signaling repressor JAZ proteins(JAZ5)were able to regulate secondary wall synthesis and lignin deposition in poplar by interacting with the secondary wall synthesis switch factor WND/MYB(WND6A/MYB3)and inhibiting the expression activation of genes related to secondary wall synthesis.The present study provides a preliminary analysis of JA regulation of secondary wall synthesis and lignin deposition in poplar.In this study,we revealed the molecular mechanism of JA regulation of secondary wall synthesis,improved the regulatory network of JA for development and stress response,provided new molecular evidence for the study of JA anabolism and regulatory mechanism in woody plants,and laid a theoretical foundation for the breeding of poplar species with excellent traits of resistance to pests and diseases.