AtPRMT5 Regulates Shoot Regeneration Through Mediating Histone H4R3 Dimethylation On KRP1 And Pre-mRNA Splicing Of RKP In Arabidopsis

Plant cells have evolved totipotency,which makes the highly differentiated tissues or organs to regenerate into new tissues or organs under special hormone conditions,accompanied by the cell fate transition,and this process is known as de novo organogenesis.De novo organ regeneration is an excellent biological system for the study of fundamental questions and the application in genetic transformation and propagation.De novo organogenesis includes two steps,and the first process is involved in the callus cells formation,while the second process includes hormone redistribution patterns establishment,stem cell initiation and organ primordium formation.The ratio of auxin and cytokinin determines the cell fates of regenerative organs.The high ratio of auxin to cytokinin promotes root formation,while the low ratio of auxin to cytokinin determines the shoot formation.Because the shoot meristem gives rise to all parts of the plant body above ground,the studies on de novo shoot regeneration have been noticed for many years.Up to now,many regulators have been identified to be involved in the shoot regeneration.The most important event is the organizing center regulator WUSCHEL(WUS)induction under auxin and cytokinin regulation.WUS protein could activate the stem cell regulator CLAVATA 3(CLV3)expression,resulting in the formation of the stem cells.Besides hormones,epigenetics,including DNA methylation,non-coding RNA,histone lysine methylation,are involved in the shoot regeneration.Protein arginine methylation is essential for diverse biological processes,including transcription modulation,flowering regulation,RNA splicing,DNA repair,and signal transduction.However,whether and how protein arginine methylation regulates shoot regeneration in vitro remains elusive.In this study,we analyzed the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis.AtPRMT5 encodes a type II PRMT that methylates proteins,including histones and RNA splicing factors.In plants,mutation of AtPRMT5 results in many developmental defects including retarded growth,darkgreen,curly leaves and delayed flowering.During shoot regeneration,functional loss of AtPRMT5 caused the decreased frequency of shoot regeneration and the reduced shoot number per callus.Chromatin immunoprecipitation analysis indicated AtPRMT5 targets KIP-RELATED PROTEIN 1(KRP1),which encodes a cyclin-dependent kinase.During shoot regeneration,the KRP1 transcript level increased in the atprmt5 mutant,which resulted from the reduced histone H4R3 methylation in the KRP1 promoter.Overexpression of KRP1 significantly reduced the frequency of shoot regeneration and shoot number per callus.Furthermore,an abnormal pre-mRNA splicing of the ubiquitin E3 ligase RELATED TO KPC1(RKP)was detected in the atprmt5 mutant.Approximately 45% of the fifth intron of RKP could not be spliced,resulting in the frameshift of RKP protein.Meanwhile,mutation of RKP inhibited shoot regeneration,implying AtPRMT5-mediated shoot formation is involved in RNA splicing of RKP.Taken together,AtPRMT5 regulated shoot regeneration through mediating histone H4R3 dimethylation on KRP1 transcription and pre-mRNA splicing of RKP.Our findings provide new insights into the function of protein arginine methylation during de novo shoot regeneration and establish a novel link between pre-mRNA splicing and shoot induction.The deep interpretation of the protein arginine methylation function would enrich the regulatory network of de novo organgenesis and provide new information for improving capacity of organ regeneration.