The Molecular Mechanism Of AtERF022 Gene Regulating Arabidopsis Root Growth

Transcription Factors Ethylene Response Factors(ERFs)are downstream components of ethylene signaling pathway,which are involved in regulating plant growth and development as well as biotic and abiotic stress responses.In this study,mutant genetics,molecular biology,transcriptomics and transgenic techniques were used to investigate the molecular mechanism of At ERF022 gene regulating plant root growth.ERF022(AT1G33760)belongs to the ERF IIIa family of Arabidopsis thaliana.Bioinformatics analysis showed that ERF022 gene was located on chromosome 1 of Arabidopsis thaliana.The total length of ERF022 gene was 1164 bp,containing only one exon but no intron.The total length of ERF022 c DNA was 555 bp,encoding 184 amino acids.qRT-PCR and RT-PCR showed the highest expression of ERF022 in leaves,followed by stem,flower,fruit shell and root.The results of protein subcellular localization showed that ERF022 was localized in the nucleus.Arabidopsis mutant erf022-1 and erf022-2 were obtained by CRISPR/Cas9 technique.The CRISPR vector was constructed,and the vector was transferred into Arabidopsis wild-type mediated by agrobacterium.Homozygous mutants were obtained through screening and identification of the plants.Sequencing results showed that the ERF022 gene in erf022-1 and erf022-2 mutants had deletion of 24 bp and 18 bp bases respectively.Overexpression vector 35::ERF022 was constructed by DNA recombination method,and transgenic complementary strains ERF022-COM1 and ERF022-COM2 and overexpressed transgenic strains ERF022-OE1 and ERF022-OE2 were screened through agrobacterium transformation and molecular identification.The root length and fresh weight of erf022-1,erf022-2,ERF022-OE1 and ERF022-OE2 growing normally on 1/2 MS medium were significantly higher than those of the wild type,and the number of lateral roots were also higher than that of the wild type,but there was no significant difference between the root hair and the wild type.According to the observation of root tip meristematic zone and elongation zone cells of each plant,the elongation zone cells of erf022-1 and erf022-2 were larger than those of the wild type,while the number of meristem cells of ERF022-OE1 and ERF022-OE2 were significantly more than that of the wild type.These results suggest that both loss of function and overexpression of ERF022 gene promote Arabidopsis thaliana root growth,but the mechanism may be different.To elucidate the mechanism of ERF022 gene in Arabidopsis thaliana root growth,we performed RNA-seq analysis.Compared with wilt type,there were 140 genes with significant difference(66 up-regulated genes,74 down-regulated genes)in erf022-1,and 229 genes with significant difference(109 up-regulated genes,120down-regulated genes)in ERF022-OE1.Through the analysis of GO and KEGG signaling pathways,the significantly different genes in erf022-1 were mainly related to signal transduction pathways,and the significantly different genes in ERF022-OE1 were mainly related to signal transduction,amino acid metabolism,and biosynthesis pathways of other secondary metabolites.By screening for pathways involved in signal transduction and secondary metabolite synthesis,we found genes that were significantly different: GASA14,LRX2,XTH19,CYP78A7,CYP96A5,ACS8,SAUR10,PME41,ABCG40,GLR1.3,PILS4,WAK3,LBD26,HB2,YUC5,GALK2,EXO,MYB73,CYP83A1,ADAP,PER33,MBP1,CYP71A13.qRT-PCR verification results showed that in erf022-1,the transcriptional levels of SAUR10,CYP83A1,GASA14,GALK2,ADAP,LRX2,XTH19,LBD26,EXO,PER33,PME41,CYP78A7,CYP96A5 and ACS8 were significantly higher than those of the wild type.The transcriptional levels of ABCG40,GLR1.3,PILS4 and WAK3 were lower than those of the wild type.In ERF022-OE1,the transcriptional levels of LRX2,CYP78A7,PILS4,LBD26,HB2,YUC5,GALK2,EXO,and MYB73 were higher than those of the wild type,while the transcriptional levels of CYP83A1,ADAP,PER33,MBP1,and CYP71A13 were lower than those of the wild type.The results of transcriptome sequencing showed that the promotion effect of ERF022 gene on root growth was closely related to auxin signal and some related transcription factors.Therefore,we analyzed the content of endogenous auxin,the synthesis and transportation of auxin and the expression of genes related to signal pathway in each plants,and carried out exogenous auxin treatment.The results showed that ERF022 gene was strongly expressed by exogenous IAA.The auxin content of erf022-1,erf022-2,ERF022-OE1 and ERF022-OE2 was higher than that of wild type.Transcriptional levels of auxin synthesizing genes TAA1,YUC1,YUC2,YUC6 and polar transport genes PIN1,PIN2,PIN3,PIN4,PIN5,PIN6,PIN7 and AUX1 in erf022-1 and erf022-2 were significantly higher than those in the wild type.The transcriptional levels of synthetic genes YUC1,YUC2,YUC6 and polar transport genes PIN1,PIN2,PIN3,PIN5,PIN6,PIN7 and AUX1 in ERF022-OE1 and ERF022-OE2 were also significantly higher than those in the wild type.In addition,the expression of the auxin response factor ARF2 gene in both ERF022 functional deletion and overexpression plants was significantly up-regulated,and the expression levels of ARF10,ARF16 and ARF19 were down-regulated.According to the above results,the molecular mechanism of ERF022 gene regulating Arabidopsis root growth was speculated:The loss of function of ERF022 gene induces the up-regulation of auxin synthesis gene,which leads to the increase of auxin content,and then the up-regulation of SAUR10(auxin reactive protein 10),which induces cell elongation.In addition,the loss of function of ERF022 gene induces the up-regulation of GASA14 gene,which promotes plant growth by promoting cell elongation in elongation region.The overexpression of ERF022 gene up-regulates the expression of YUC5(auxin synthesis gene)and down-regulates the expression of CYP83A1(cytochrome P450-83A1)gene,which leads to the increase of auxin content,and then induces the expression of LBD26(including LOB domain protein 26)through auxin signal to regulate lateral root formation.In addition,ERF022 overexpression induced upregulation of HB2(leucine homologue 2)gene,which encodes a developmental regulator involved in cell proliferation.In conclusion,functional loss and overexpression of ERF022 gene promote auxin biosynthesis and transport through different pathways,induce the expression of SAUR10,GASA14,LBD26 and HB2 genes,regulate the elongation and proliferation of root cells,and thus promote root growth.