PtrJMJ25 Is Involved In The Regulation Of Anthocyanin Biosynthesis In Poplar

Anthocyanins are important secondary metabolites in plants and are mainly found in epidermal cells of the petals,fruits,stems and leaves of plants.In plants,anthocyanins can determine the color of flowers and fruits,as well as resist to environmental stress.At the same time,anthocyanins can attract insects to pollinate and remove free radicals produced by plant cells.Therefore,to understand the regulation mechanism of anthocyanin biosynthesis,not only could improve our knowledge about how plants adapt to environmental stresses,but also helpful for taking advantages of plant resources.Thus,it has important scientific significance and industrial prospect in production and practice.Recently,it has been reported that the JMJC histone demethylase family member IBM1 in Arabidopsis thaliana,participated in regulation of anthocyanin biosynthesis through modifying methylated histone and releasing transcription.As a genetic model in woody plants,poplar is the most widely grown fast-growing tree in the world and is widely used in the study of important secondary metabolites of woody plants.However,whether histone methylation is involved in regulation of anthocyanin biosynthesis in poplar is not been reported yet.Therefore,it is helpful for improve our understanding in mechanism of anthocyanin biosynthesis by studying the epigenetic regulation profiles in poplar.First,we identified 26 members of the JMJC family proteins in the poplar genome using bioinformatics analysis.6 of them were homologous ofIBM1 and belong to the JHDM2 subfamily,which are considered to be H3K9-specific histone demethylase.Since the IBM1 is involved in the regulation of anthocyanin biosynthesis through regulating the light signal transduction pathway,these IBM1 homologous were checked on their response to light and darkness in wild-type poplar.PtrJMJ25 was identified by its significantly elevated expression in darkness and constant level in light.By constructing a promoter expression vector and determine the GUS enzyme activity,it showed that the transcription of PtrJMJ25 was increased along with the time of dark.These results indicate that PtrJMJ25 is induced under dark condition.The results are shown as follows:(1)Expression of PtrJMJ25 in ibm1 mutant could restore the phenotype of mutant plants such as smaller,narrower leaves and pollen abortion caused by loss of IBM1,indicating that PtrJMJ25 is highly conserved with IBM1 in their function on plants development.(2)By genetic transformation,we had obtained transgenic poplars that overexpression of PtrJMJ25.As cultured in outdoor light,the wild-type poplar exhibited obvious anthocyanin accumulation in the leaves,while the transgenic poplars had no significant anthocyanin accumulation.At the molecular level,the key enzyme genes in anthocyanin biosynthesis such as DFR,ANS,and UFGT were significantly down-regulated in overexpression of PtrJMJ25 compared to the wild type,demonstrating that PtrJMJ25 is involved in the repression of anthocyanin synthesis in poplar.(3)In contrast to IBM1 directly control SPAs transcription,overexpression of PtrJMJ25 did not change the expression of SPAs homologues in poplar.It indicates that PtrJMJ25 did not affect anthocyanins through regulating the expression of SPAs homologues in poplar.(4)In PtrJMJ25-overexpressing lines,the expression of a known anthocyanin inhibitor in poplars,MYB182,was significantly up-regulated,and the expression of MYB182 was also able to respond to darkness induction,suggesting that the functional relationship between PtrJMJ25 and MYB182 in inhibiting anthocyanin synthesis.(5)The CHIP assay demonstrated that PtrJMJ25 could directly bind to the chromatin of MYB182 in poplar cells.Overexpression of PtrJMJ25 resulted in a decreased level of H3K9 demethylation on the chromatin of MYB182 and a decrease of non-CG DNA methylation levels on MYB182 locus.These results demonstrate that PtrJMJ25 can inhibit anthocyanin synthesis by directly releasing the expression of MYB182.(6)Using the CRISPR-CAS9 mediated genome edit,we obtained the transgenic poplar lines with knocked-out endogenous PtrJMJ25.Under dark condition,both of the anthocyanins content and the expression levels of anthocyanin synthesis genes in leaves of PtrJMJ25 knockout plants were increased compared to the wild type,while the expression level of MYB182 was significantly decreased.These results indicate thatthe activation of MYB182 by PtrJMJ25 in wild-type poplars is necessary for turn off the synthesis of anthocyanins in the dark.In summary,we proved that PtrJMJ25,a histone demethylase in poplar,could promote the expression of MYB182 through directly modulating the histone methylation on the chromatin in response to darkness,and further control anthocyanin biosynthesis inner cells.This study reveals the function and mechanism of epigenetic modification in the synthesis of anthocyanin in woody plants,and improved our understanding on how plant anthocyanin synthesis accurately responds to environmental change.