Transactivation Targets And Ubiquitin Modification Analysis Of Opaque2 In Maize

Maize(Zea mays)has made outstanding contribution to the human civilization.Today,maize is still one of the most important crops.Kernel is the nutrient storage organ of maize for its abundance in starch and protein.The nutrient value of maize kernel highly depends on the quality of its storage proteins.Traditional maize seed has low protein quality because it lacks two of the essential amino-acids,lysine and tryptophan.Opaque2 encodes a transcription factor exclusively expressed in endosperm.The opaque2(o2)mutant is an important genetic resource for maize quality protein breeding due to the significantly improved essential amino acids content in its storage proteins.But o2 also brings some inferior agronomic traits that discouraged its broad application in breeding programs.Functional analysis of O2 encourages the clarification of the inferior agronomic traits.To clarify the molecular mechanism of the inferior agronomic traits in o2 mutant,the downstream regulation targets of O2 were identified.We studied the transcriptome of o2 mutant using RNA Sequencing(RNA-Seq)and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing(ChIP-Seq).The RNA-Seq analysis revealed 1,065 differentially expressed genes(DEGs)and 383 differentially expressed lnc RNAs.The DEGs cover a wide range of functions related to nutrient reservoir activity,nitrogen metabolism,stress resistance,etc.ChIP-Seq analysis detected 1,686 O2 DNA binding sites distributed over 1,143 genes.Overlay of the RNA-Seq and Ch IP-Seq results revealed 35 O2-modulated target genes.We identified 4 new O2 binding motifs;among them,TGACGTGG appears to be the most conserved and strongest.We confirmed that,except for the 16 kDa and 18 kDa zeins,O2 directly regulates expression of all other zeins.O2 directly regulates two transcription factors,genes linked to carbon and amino acid metabolism and abiotic stress resistance.We built a hierarchical regulatory model for O2 that provides understanding of its pleiotropic biological effects.Post-translational modifications(PTMs)of proteins enable fast modulation of protein function in response to metabolic and environmental changes.To study the PTMs of O2,we identified several ubiquitin E3 ligase candidates for O2 by analyzing our previous yeast two hybridization results using O2 protein as a bait.A17 is one of the candidates and it is a C3HC4 RING domain and WD40/YVTN repeat domain containing protein.In vitro and in vivo studies showed that O2 has interaction with A17.Also,we proved that A17 has E3 activity and can mono-ubiquitinate O2 in vivo.Mono-ubiquitination of cytoplasmic O2 promotes its nuclear localization and thus enhances its transactivation on the target promoters.By RNA-seq and Ch IP-Seq,we confirmed 35 genes directly regulated by O2.Functional study of these genes suggests that O2 not only directly activates genes associated with a variety of nutrient storage genes and enzymes involved in carbon and amino acid metabolism pathways,but also controls transcription factors that are,in turn,regulating various other aspects of plant metabolism.The study of E3 ligase protein interacting with O2 suggests that O2 can be mono-ubiquitinated in vivo.The study on O2’s downstream transcription regulation framework and its upstream post-translational modification will help to expand the O2 based regulation network and provide theoretical basis for quality protein maize breeding.