HUB1 Regulates Apical Hook Development And Screens Transcription Factors Of Its Target Modifier Gene TSB1

Our laboratory previously screened the Arabidopsis mutant ckrw2(CYTOKININ INDUCED ROOT WAVING 2)through forward genetics screening.ckrw2 showed auxin-deficient phenotypes such as short main root and loss of gravitational tropism.The above phenotypes were found to be related to the loss of function of the gene HUB1(HISTONE MONOUBIQUITINATION 1)through gene cloning.HUB1 encodes an E3 ubiquitin ligase responsible for catalyzing the monoubiquitination of histone H2 B.The laboratory’s previous CHIP experiments proved that HUB1 regulates its gene expression by directly affecting the monoubiquitination modification level of histone H2 B entangled by the auxin synthesis genes TSB1,WEI7,AMI1,and YUC7.In this study,the mechanism of HUB1 participating in dark morphogenesis was analyzed based on the phenotype of apical hook defect in hub1/2 mutant,and how HUB1 targetedly regulated TSB1 gene was discussed.A previous study found that hub1/2 mutants exhibited a phenotype of defective apical hook development when grown in the dark.To further investigate whether the phenotype of defective apical hook development in hub1/2 mutants is related to the decreased levels of endogenous auxin caused by decreased transcription levels of the genes TSB1,WEI7,AMI1,and YUC7,we first examined the tissue expression patterns of HUB1 and changes in DR5::GUS chemical staining at the apical hook of hub1 mutants in dark.The results showed that HUB1 was expressed both inside and outside of the apical hook,and the asymmetric distribution of auxin at the apical hook of the hub1 mutant was lower than that of the wild type.In the subsequent study,we found that the transcription levels of WEI7 and TSB1 in hub1 mutant decreased,but the morphogenesis defect phenotype of hub1 mutant was stronger than that of wei7 and trp2(allelic mutation of TSB1 gene)mutants,suggesting that HUB1 may also have other functions in the development of apical hook.Among the interaction proteins screened in previous studies with HUB1,three potential proteins,EBF1(Ethylene Binding F-box Protein 1),IAA32 and IAA34,were also involved in the development of hooks in dark morphogenesis.Since EBF is involved in ethylene signaling,we treated hub1 / 2 mutant with ACC,the precursor of ethylene synthesis,and found that the phenotype of apical hook deletion in hub1 mutant was not sensitive to ACC treatment,suggesting that HUB1 may regulate apical hook development through ethylene signaling.At the same time,the interaction between HUB1 and auxin IAA32 protein was re-verified by yeast two-hybrid,and the interaction between HUB1 and IAA32 was further verified by pull-down experiment.Based on the above results,we speculate that the apical hook deficient phenotype caused by the deletion of H2Bub1 in Arabidopsis is related to the reduction of endogenous auxin content in the plant,and it is not ruled out that other pathways,such as IAA32/34 mediated auxin signaling pathway are involved in development of the apical hook in dark morphogenesis and so on.In addition,in order to further study how HUB1 targets and regulates the transcription of TSB1 gene,we screened the transcription factors of TSB1 gene by yeast one-hybrid,namely DIL9-3,RAP2.2,ERF5,Histone1.1,CCR2,HON5.We speculate that these transcription factors may participate in the transcriptional regulation of TSB1 by recruiting HUB1.Unfortunately,the yeast two-hybrid results showed that none of the transcription factors DIL9-3,ERF5,and RAP2.2 could interact with HUB1 protein,indicating that all three transcription factors were cannot recruit HUB1 protein to TSB1 target genes.