Function And Mechanism Analysis Of Histone Demethylase PtrJMJ11 In Secondary Development In Populus Tomentosa

Vascular system plays as vital structure during plant development upon its abilities on water and nutrition transportation.Stem vascular system is composed of inner xylem and outer phloem and strictly subject to multiple regulations.Epigenetic modification is a strategy which doesn’t change DNA sequences while regulating gene expression and participates numerous biological processes,which has been reported.The H3K9 methylation,as a vital type of epigentical modification,occurs at the ninth lysine of histone H3 and cooperates with DNA methylation to regulate multiple developmental and metabolic processes in plants.However,whether H3K9 methylation is involved in the regulation of plant stem vascular development remains unclear yet.Hence,our investigation into whether histone methylation functions in plants stem vascular system development is helpful to perceive epigenetic regulation mechanism of it.In Arabidopsis,it has proved that proteins belong to Jmj C family are responsible for histone demethylation.But it is still confused about participation of them in regulating plants stem vascular system.In this study,we identified and characterized a phloem-specific JHDM2 subfamily histone demethylase,PtrJMJ11 in poplar.To investigate the function and mechanism of PtrJMJ11 in development of stem vascular system,a series of experiments and analysis were preformed as follow:1.We performed RNA sequencing using the continuously different internodes from wild type poplar.As a result,among all 26 of Jmj C family members in poplar,PtrJMJ11 exhibits most high expression levels during each internode that represent different developmental stages.That implies the possible role of PtrJMJ11 in the regulation of plant stem vascular development.2.By in situ and GUS-histological staining,PtrJMJ11 was found specially expressed in phloem cells during the development of stem vascular in poplar.3.Through in vitro analysis about the activity of histone demethylase,it revealed that PtrJMJ11 was capable to demethylate H3K9me1 and H3K9me2.This result is further confirmed inner plant cells in which the expression of PtrJMJ11 decreased H3K9me2 and H3K9me1 levels dramatically and specifically.4.By using histological observation and scanning electron microscope,we found that the division and differentiation of cambial cells were both decrease in ptrjmj11 mutant,as well as the differentiation of phloem was declined.Opposite phenotypes were found in the PtrJMJ11-overexpression plants.5.A RNA deep sequencing was performed across the wild type,ptrjmj11 and PtrJMJ11-overepression poplars,using the eighth internodes of stem.Based on the Gotology(GO)analysis,the DEGs(Different expression genes)were mainly enriched in catalytic and binding strategies.RT-qPCR further revealed that,the genes related with division and differentiation of stem cambium were dis-regulated accordingly in ptrjmj11 mutant and PtrJMJ11-overexpression plants.Among these genes,the expression of Ptr CLE41a/b,members of poplar CLAVATA3/Embryo Surrounding Region-related(CLE)gene family,were significantly regulated by PtrJMJ11.6.In situ analysis revealed that Ptr CLE41a/b was specially expressed in phloem in the stem of poplar.Through protein sequences alignment,a 12 amino acid oligopeptide TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR(TDIF)display same sequences between poplar and Arabidopsis.The deficiency of cambium division and differentiation resulted from ptrjmj11 mutant could be rescued to a WT-like phenotype by supplementing exogenous TDIF.7.By Chromatin immunoprecipitation-qPCR(ChIP-qPCR)assays,we found that PtrJMJ11 could directly associate with the chromatin of Ptr CLE41a/b.Loss or overexpresison of PtrJMJ11 cause increased or reduced the occupancy of H3K9me2 and H3K9me1 at the Ptr CLE41a/b locus,whereas the H3K9me2/1 levels at Ptr CLE41 c locus change only modestly.To sum up,phloem specially-expressed gene PtrJMJ11 could directly target to Ptr CLE41a/b and substantially demethylate H3K9 on its chromatin,and then,releases the expression of Ptr CLE41a/b specifically in phloem to promote both the division and differentiation of vascular cambial cells in poplar stem development.This study revealed the function and mechanism of PtrJMJ11 in epigenetic regulation of vascular development and improved our understanding in genetic control and modification of wood production in trees.